Multispecific antibodies

ABSTRACT

The present invention relates to heterodimerically-tethered bispecific protein complexes (according to the general formula of A-X:Y-B) and libraries/multiplexes thereof for use in research and therapy and in particular an in vitro/ex vivo method of detecting synergistic biological function of otherwise unknown pairs of targets.

FIELD OF INVENTION

The present disclosure relates to a method, in particular an in vitro/exvivo method, of detecting synergistic biological function in aheterodimerically-tethered bispecific protein complex,libraries/multiplexes of the bispecific protein complexes, and kits andcompositions thereof. The disclosure further relates to said novelbispecific protein complexes and use of the same in therapy, researchand experimental purposes (in particular in assays looking forsynergistic biological function). The present disclosure also extends tomethods of preparing said bispecific complexes.

BACKGROUND OF INVENTION

Biological mechanisms in vivo are extremely complicated cascades ofsignals, which are difficult to deconvolute and understand. Activationof T cells requires at least two signals. The recognition of the antigenby the T cell receptor is considered the first signal and the secondsignal arises from co-stimulation which results from the ligation ofadditional surface molecules on the T cell with additional molecules onan antigen presenting cell.

Thus T cell activation can be used to illustrate that the modulation ofbiological functions can require multiple signals. Other biologicalprocesses are equally complicated or more complicated. Whilst in vitroscreening based on cells has and can assist with gaining insights intoin vivo mechanisms the problem still arises of how to identifyappropriate ligand pairs which modulate the biological function.

Bispecific antibodies are widely expected to play a major role in thenext generation of biotherapeutics (D. Holmes, Nature Rev Drug DiscNovember 2011:10; 798). They have the potential to deliver superior,long term, broad efficacy in a greater proportion of patients. This canbe achieved by either co-engaging different antigens simultaneouslywithin a common disease pathway, thereby reducing redundancy; or bytargeting antigens from independent pathways to provide an additive orsynergistic effect.

Bispecific antibodies facilitate access to novel biology such as:

-   -   1) cross-linking receptors on a cell,    -   2) inducing cell mediated effects,    -   3) localizing a cytokine to a cell to regulate signaling or        locally block cytokine function,    -   4) engaging multiple epitopes simultaneously to generate “new        activity”, increase function or specificity, which may not be        exhibited by a single monoclonal antibody or indeed mixtures of        un-linked antibodies (‘poly-monoclonals’).

Current strategies to engage dual targets are largely based on rationaldesign of known mechanisms and include: cross-linking inhibitoryreceptors, co-engagement/clustering of receptors, blocking multiplestimulatory pathways, selective engagement of inhibitory receptors andblocking distinct pathways such as co-stimulation & cytokine signaling.However, the current state of the art in relation to known mechanismsand targets is a limiting factor to progress in this area.

Whilst bispecific antibodies have enormous potential as biologicaltherapeutics they also present an increased set of challenges withindiscovery and development compared to monoclonal antibodies. Two keyareas of difficulty are, 1) the development of a successful bispecificantibody format, and 2) selecting the pairs of targets to which thebispecific antibody will crosslink or co-engage.

Many promising bispecific antibody formats have now been developed thatcould potentially work as successful therapeutics including DVD-Ig(Abbvie), DuoBodies (Genmab), Knobs-in-Holes (Genentech), Common lightchain (Merus). However, in each of these cases these formats are notideally suited to high throughput target-dual-antigen discoveryscreening to enable the discovery of novel antigen pairs forcrosslinking with bispecific antibodies. Typically for a singlebispecific antibody construct at least two variable regions need to besub-cloned from the original source of discovery vectors (e.g. phagedisplay, hybridoma or single B-cell cloning) into appropriate bispecificexpression vectors, each arm of the bispecific has to be expressed andthe resulting bispecific antibody purified. This cloning and subsequentexpression effort quickly becomes a significant practical bottleneck iflarge numbers of pairs of variable regions are to be combined in anattempt to screen for the most efficacious combination of discoveredvariable regions or to discover novel antigen pairs. For example, if 50unique antibodies are discovered against a panel of 50 cell surfacetargets, then a total of 2500 bispecific antibodies could potentially begenerated (envisaged as an X-by-Y grid). With the bispecific antibodyformats described above this would require at least 100 individualcloning reactions (50-X and 50-Y) followed by 2500 antibody expressionexperiments. Increasing the number of starting monoclonal antibodies to100 would increase the minimal number of cloning reactions to 200 (100-Xand 100-Y) and the expression number to 10,000.

Generally the root cause of this ‘expression bottleneck’ is the factthat the formats described above require both protein chain ‘halves’ ofthe final bispecific construct to be expressed simultaneously within asingle expression experiment in the same cell. Therefore, for manyformats, to produce 2500 bispecific antibodies, 2500 expressionexperiments are required.

The ‘expression bottleneck’ is further exacerbated if the bispecificantibody format is monocistronic (i.e. cloned and expressed as a singlechain protein), for example single chain diabodies, as the number ofcloning experiments would be 2500 and 10,000 respectively for thenumbers given above.

Furthermore after expression, extensive purification may be required toisolate the desired construct.

Some bispecific approaches employ a common light chain in the bispecificconstructs in order to reduce the amount of cloning, although thisdoesn't reduce the number of expression experiments. Furthermore, usinga common chain, such as a common light chain, makes the challenge ofantibody discovery harder as it is more difficult to find the startingantibody variable domains as the antibody needs to bind its antigen witha high enough affinity through one chain, such as the heavy chain,alone.

Accordingly the use of current bispecific formats in large scale andhigh throughput screening to identify novel antigen pairs is impracticaland has led to the continued use of solely hypothesis driven approachesto bispecific antigen targeting.

We propose that rather than designing and testing a limited selection ofbispecific antibodies that engage given epitopes on two known targets,the true potential of exploiting access to novel biology with bispecificantibodies can only be achieved through a broad functional screeningeffort with a large, diverse combinatorial panel of bispecificantibodies or protein ligands. To facilitate this screening a format anda method is required that enables the generation of large numbers ofdiverse bispecific proteins which can be readily constructed andscreened for functional effects in a variety of functional screens. Thisapproach allows for the serendipitous identification of synergisticpairs.

Thus it would be useful to generate and screen a large number ofbispecific protein complexes present as combinations of various antigenspecificities. In particular, it would be useful to be able to generateand screen a large number of different bispecific antibody complexes ina quick and efficient manner. There are a range of existing methods formanufacturing bispecific antibodies as already described above. However,each of these methods has its disadvantages, as do alternative methodsas further described in more detail below.

The problem of how to efficiently identify targets for bispecific andmultispecific constructs has not been adequately addressed in the art.For example WO2014/001326 employs chemical conjugation of a protein to aDNA fragment, wherein the DNA fragment hybridises to a complementary DNAsequence that links two such proteins together for generatingtailor-made patient-specific multispecific molecules comprising at leasttwo targeting entities. There are number of difficulties associated withthis approach if it were to be applied to identifying new bispecificcombinations, for example conjugation of the protein to the DNA canresult in damage to the activity and/or structure of the protein. Inparticular protein-DNA hybrids are not naturally occurring thus there isa potential for interference. In addition the chemical conjugationrequired joining the protein and DNA adds complexity, time and expenseto the process.

Coupling and conjugation techniques exist for generating antibody drugconjugates and in vivo targeting technologies. Traditional chemicalcross-linking is labour intensive as the relevant species may need to bepurified from homodimers and other undesirable by-products. In addition,the chemical modification steps can alter the integrity of the proteins,thus leading to poor stability or altered biological function. As aresult, the production of bispecific antibodies by chemicalcross-linking is often inefficient and can also lead to a loss ofantibody activity.

Another method of manufacturing bispecific antibodies is by cell-fusion(e.g. hybrid hybridomas), wherein the engineered cells express two heavyand two light antibody chains that assemble randomly. Since there are 4possible variants to choose from, this results in the generation of 10possible bispecific antibody combinations, of which only some (in manycases, only one) combinations would be desired. Hence, generatingbispecific antibodies by cell-fusion results in low production yieldsand also requires an additional purification step in order to isolatethe desired bispecific antibodies from the other bispecific antibodiesproduced. These disadvantages increase manufacturing time and costs.

Recombinant DNA techniques have also been employed for generatingbispecific antibodies. For example, recombinant DNA techniques have alsobeen used to generate ‘knob into hole’ bispecific antibodies. The ‘knobinto hole’ technique involves engineering sterically complementarymutations in multimerization domains at the CH3 domain interface (seee.g., Ridgway et al., Protein Eng. 9:617-621 (1996); Merchant et al.,Nat. Biotechnol. 16(7): 677-81 (1998); see also U.S. Pat. Nos. 5,731,168and 7,183,076). One constraint of this strategy is that the light chainsof the two parent antibodies have to be identical to prevent mispairingand formation of undesired and/or inactive molecules when expressed inthe same cell. Each bispecific (heavy and light chains thereof) must beexpressed in a single cell and the protein product generally containsabout 20% of homodimer, which is subsequently removed by purification.

Other approaches are based on the natural exchange of chains infull-length IgG4 molecules (Genmab Duobody). However, this approach alsohas difficulties because it does not allow a construct to be preparedwithout an Fc region. As the Fc region can contribute to biologicalactivity it may be difficult to establish if an activity observed isbased on the combination of variable regions, the Fc or both inbispecific molecules comprising an Fc. Furthermore, the exchange is adynamic process and this may lead to difficulties in relation to whatthe entity tested actually is.

Thus there is a need for new methods of generating bispecific proteincomplexes to enable the more efficient and higher throughput screeningof bispecific antibodies. In particular, there is a need for a formatand a method wherein a selection of any two antibodies or antibodyfragments from a pool of available antibodies or antibody fragments canbe readily combined to efficiently produce a multiplex of differentbispecific antibodies, whilst, for example avoiding or minimising theformation of homodimers. Assembling different bispecific antibodiesefficiently is particularly important when screening for synergisticbiological function for new combinations of antigen specificities, inparticular where heterodimers are essential for discovering thatfunction.

SUMMARY OF INVENTION

In one aspect there is provided a new bispecific format particularlysuitable for use in screening because all of the components can beexpressed from a cell as individual units, essentially withoutaggregation and the units can be assembled simply by mixing withoutemploying conjugation or coupling chemistry and with minimalhomodimerisation.

Thus there is provided a bispecific protein complex having the formulaA-X:Y-B wherein:

-   -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   : is a binding interaction between X and Y;    -   A is a first protein component of the bispecific protein complex        independently selected from the group comprising a Fab fragment,        a Fab′ fragment, sdAb, and a single chain Fv (scFv);    -   B is a single chain Fv or sdAb;    -   X is a first binding partner of a binding pair independently        selected from an antigen, a Fab fragment, a Fab′ fragment, a        single chain Fv and a sdAb; and    -   Y is a second binding partner of the binding pair independently        selected from antigen, a Fab fragment, a Fab′ fragment, a single        chain Fv and a sdAb;    -   with the proviso that when X is an antigen Y is a Fab fragment,        a Fab′ fragment, a single chain Fv or a sdAb specific to the        antigen represented by X and when Y is an antigen X is a Fab        fragment, a Fab′ fragment, a single chain Fv or a sdAb specific        to the antigen represented by Y.

X and Y may be fused to A and B, respectively, either at the N-terminalor at the C-terminal of A and B.

Within the present disclosure, the fusion proteins' terms “A-X” and“Y-B” may be analogously indicated as “X-A” or “B-Y”. The same appliesto the term for the heterodimeric-tether “X:Y” which can also beindicated herein as “Y:X”.

In one example there is provided a bispecific protein complex having theformula A-X:Y-B wherein:

-   -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   : is a binding interaction between X and Y;    -   A is a first protein component of the bispecific protein complex        independently selected from the group comprising a Fab fragment,        a Fab′ fragment, a sdAb and a single chain Fv (scFv);    -   B is a single chain Fv or sdAb;    -   X is a first binding partner of a binding pair independently        selected from an antigen, a Fab fragment, a Fab′ fragment, a        single chain Fv and sdAb; and    -   Y is a second binding partner of the binding pair independently        selected from antigen, a    -   Fab fragment, a Fab′ fragment, a single chain Fv and a sdAb;    -   with the proviso that when X is an antigen Y is a Fab fragment,        a Fab′ fragment, a single chain Fv or a sdAb specific to the        antigen represented by X and when Y is an antigen X is a Fab        fragment, a Fab′ fragment, a single chain Fv or a sdAb specific        to the antigen represented by Y.

In one embodiment X is fused, optionally via a linker, to the C-terminalof a scFv or the C-terminal of the heavy chain in the Fab fragment orFab′ fragment, whichever is represented by A.

In one embodiment Y is fused, optionally via a linker, to the C-terminalof the scFv represented by B.

In one embodiment X is fused, optionally via a linker, to the N-terminalof a scFv or the N-terminal of the heavy chain in the Fab fragment orFab′ fragment, whichever is represented by A.

In one embodiment Y is fused, optionally via a linker, to the N-terminalof the scFv represented by B.

In one embodiment the variable X or Y is a Fab fragment, a Fab′fragment, a scFv, or sdAb and the other variable (X or Y) is a peptide.

When X or Y is a Fab or Fab′ molecule the C terminal of the fragment,such as the C-terminus of the heavy chain CH1 or the light chain CL,will generally be connected via a linker to the C terminal of theantibody fragment A or B.

In one embodiment the binding affinity between X and Y is 5 nM orstronger, for example 900 pM or stronger, such as 800, 700, 600, 500,400 or 300 pM.

In one embodiment at least one (such as one) of A, X and/or Y is a Fabor Fab′ molecule. Advantageously having at least one Fab or Fab′molecule in the format is beneficial to the stability of the format, forexample physical stability and may minimise aggregation or similarundesirable effects that may affect the format, especially in theabsence of the Fab or Fab′ fragment.

In one embodiment the bispecific complex of the disclosure comprisesonly one Fab fragment or only one Fab′ fragment.

In one embodiment the bispecific complex of the disclosure comprises nomore than one or no more than two scFvs.

In one embodiment A is a Fab or Fab′ fragment, such as a Fab fragment.

In one embodiment A is a scFv.

In one embodiment A is a sdAb.

In one embodiment A is a scFv and X or Y is a Fab or Fab′ fragment.

In one embodiment B is a scFv.

In one embodiment B is a sdAb.

In one embodiment B is a scFv and X or Y is a Fab or Fab′ fragmentFormats which incorporate one or more scFvs as A and/or B are useful forscreening because it allows scFv molecules from libraries, such as phagelibraries, to be screened rapidly without the need to reformat intoother antibody fragments, such as a Fab.

In one embodiment (in particular where A is a Fab, Fab′) X isindependently selected from scFv, a sdAb and a peptide, with the provisothat when X is Fab, Fab′, a scFv or sdAb then Y is an antigen, such as apeptide, and when X is a peptide Y is a Fab fragment, a Fab′ fragment, ascFv or a sdAb.

In one embodiment (in particular where A is a scFv) X is independentlyselected from, a Fab fragment, Fab′ fragment and a peptide, with theproviso that when X is a Fab or Fab′ fragment then Y is an antigen, suchas a peptide, and when X is a peptide Y is a Fab fragment, a Fab′fragment.

In one embodiment X is a Fab fragment or a Fab′ fragment, such as a Fabfragment.

In one embodiment X is a scFv.

In one embodiment X is a sdAb.

In one embodiment Y is a Fab fragment or a Fab′ fragment, such as a Fabfragment.

In one embodiment Y is a scFv.

In one embodiment Y is a sdAb.

In one embodiment B is a scFv and Y is a Fab or Fab′ fragment.

In one embodiment X is a peptide.

In one embodiment Y is a peptide.

In one embodiment the peptide of X or Y is in the range of 5 to 25 aminoacids in length, in particular a peptide GCN4, a variant, a derivativeor a fragment thereof.

In one embodiment wherein X or Y represents a Fab fragment, a Fab′fragment, a scFv or a sdAb specific to the peptide GCN4 (SEQ ID NO:1 oramino acids 1 to 38 of SEQ ID NO:1), such as the scFv 52SR4 (SEQ IDNOs:3, 98 or 99 or amino acids 1 to 243 of SEQ ID NO:3). Where X or Y isa Fab or Fab′ fragment which binds GCN4 it may comprise the VH and VLregions from scFv 52SR4. Clearly when X or Y is a Fab fragment, a Fab′fragment, a scFv or a sdAbis specific to the peptide GCN4, a variant, aderivative or fragment thereof (SEQ ID NO:1 or amino acids 1 to 38 ofSEQ ID NO:1 in Table 1A, wherein the amino acids in bold are optionaland the amino acids in italics are the sequence of the linker), then thecorresponding variable X or Y needs to the corresponding GCN4 peptide ora variant, derivative or fragment thereof, such as amino acids 1 to 38of SEQ ID NO:1 or part thereof. The nucleotide sequence encoding theGCN4 peptide according to SEQ ID NO: 1 is shown in SEQ ID NO: 1 as SEQID NO: 2.

TABLE 1A GCN4 (7P14P) ASGGGRMKQLEPKVEELLPKNYHLENEVARLKKLVGERHHHHHHSEQ ID NO: 1 GCN4 (7P14P)GCTAGCGGAGGCGGAAGAATGAAACAACTTGAACCCAAGGTTGAAGAATTGCTT SEQ ID NO: 2CCGAAAAATTATCACTTGGAAAATGAGGTTGCCAGATTAAAGAAATTAGTTGGCGAACGCCATCACCATCACCATCAC 52SR4 dsDAVVTQESALTSSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTN scFvNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWYSDHWVFGCGTKLTV SEQ ID NO: 3LGGGGGSGGGGSGGGGSGGGGSDVQLQQSGPGLVAPSQSLSITCTVSGFLLTDYGVNWVRQSPGKCLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSSAAAHHHHHHEQKLISEEDL 52SR4 dsGATGCGGTGGTGACCCAGGAAAGCGCGCTGACCAGCAGCCCGGGCGAAACCGTG scFvACCCTGACCTGCCGCAGCAGCACCGGCGCGGTGACCACCAGCAACTATGCGAGC SEQ ID NO: 4TGGGTGCAGGAAAAACCGGATCATCTGTTTACCGGCCTGATTGGCGGCACCAACAACCGCGCGCCGGGCGTGCCGGCGCGCTTTAGCGGCAGCCTGATTGGCGATAAAGCGGCGCTGACCATTACCGGCGCGCAGACCGAAGATGAAGCGATTTATTTTTGCGTGCTGTGGTATAGCGACCATTGGGTGTTTGGCTGCGGCACCAAACTGACCGTGCTGGGTGGAGGCGGTGGCTCAGGCGGAGGTGGCTCAGGCGGTGGCGGGTCTGGCGGCGGCGGCAGCGATGTGCAGCTGCAGCAGAGCGGCCCGGGCCTGGTGGCGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTCTCCTGACCGATTATGGCGTGAACTGGGTGCGCCAGAGCCCGGGCAAATGCCTGGAATGGCTGGGCGTGATTTGGGGCGATGGCATTACCGATTATAACAGCGCGCTGAAAAGCCGCCTGAGCGTGACCAAAGATAACAGCAAAAGCCAGGTGTTTCTGAAAATGAACAGCCTGCAGAGCGGCGATAGCGCGCGCTATTATTGCGTGACCGGCCTGTTTGATTATTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGCGGCCGCCCATCACCATCACCATCACGAACAGAAACTGATTAGCGAAGAAGATCTGTAATAG SEQ ID NO: 98DAVVTQESALTSSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWYSDHWVFGCGTKLTVLGGGGGSGGGGSGGGGSGGGGSDVQLQQSGPGLVAPSQSLSITCTVSGFLLTDYGVNWVRQSPGKCLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSS SEQ ID NO: 99DVQLQQSGPGLVAPSQSLSITCTVSGFLLTDYGVNWVRQSPGKCLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSSPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWYSDHWVFGCGTKLTVLGGGGGSGGGGSGGGGSGGGGSDAVVTQESALTSSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWYSDHWVFGCGTKLTVL SEQ ID NO: MSVPTQVLGLLLLWLTDARC 100 SEQ ID NO:MEWSWVFLFFLSVTTGVHS 101 SEQ ID NO: MDWLWTLLFLMAAAQSAQA 102 SEQ ID NO:MGWSWTFLFLLSGTSGVLS 103

Other variants of the GCN4 peptides are shown in Table 1B (SEQ ID NO:75-97), wherein the amino acids in bold are optional and the amino acidsin italics form the sequence of the linker. It should be noted thatdespite variants according to sequences shown in SEQ ID NOs: 75 to 82comprise a linker of a repetition for four times of four glycineresidues and one serine (G4S), variants with linkers shorter (1×G4S,2×G4S or 3×G4S) or longer (5× G4S etc.) are also contemplated herein).

TABLE 1B SEQ ID NO: 75 GGGGSGGGGSGGGGSGGGGSYHLENEVARLKKLVGERHHHHHHSEQ ID NO: 76 GGGGSGGGGSGGGGSGGGGSYHLENEVARLKALVGERHHHHHH SEQ ID NO: 77GGGGSGGGGSGGGGSGGGGSYHLENEVARLAKLVGERHHHHHH SEQ ID NO: 78GGGGSGGGGSGGGGSGGGGSYHLENEVARLQKLVGERHHHHHH SEQ ID NO: 79GGGGSGGGGSGGGGSGGGGSYHLENEVARLNKLVGERHHHHHH SEQ ID NO: 80GGGGSGGGGSGGGGSGGGGSYHLENEVARLAALVGERHHHHHH SEQ ID NO: 81GGGGSGGGGSGGGGSGGGGSYHLENEVARLQALVGERHHHHHH SEQ ID NO: 82GGGGSGGGGSGGGGSGGGGSYHLENEVARLNALVGERHHHHHH SEQ ID NO: 83ASGGGAMKQLEPKVEELLPKNYHLENEVARLKKLVGERHHHHHH SEQ ID NO: 84ASGGGRMKQLEPKVEELLPKNYHLENEVARLKALVGERHHHHHH SEQ ID NO: 85ASGGGAMKQLEPKVEELLPKNYHLENEVARLKALVGERHHHHHH SEQ ID NO: 86ASGGGRMKQLEPKVEELLPKNYHLENEVARLAKLVGERHHHHHH SEQ ID NO: 87ASGGGRMKQLEPKVEELLPKNYHLENEVARLQKLVGERHHHHHH SEQ ID NO: 88ASGGGRMKQLEPKVEELLPKNYHLENEVARLNKLVGERHHHHHH SEQ ID NO: 89ASGGGAMKQLEPKVEELLPKNYHLENEVARLAKLVGERHHHHHH SEQ ID NO: 90ASGGGAMKQLEPKVEELLPKNYHLENEVARLQKLVGERHHHHHH SEQ ID NO: 91ASGGGAMKQLEPKVEELLPKNYHLENEVARLNKLVGERHHHHHH SEQ ID NO: 92ASGGGRMKQLEPKVEELLPKNYHLENEVARLAALVGERHHHHHH SEQ ID NO: 93ASGGGRMKQLEPKVEELLPKNYHLENEVARLQALVGERHHHHHH SEQ ID NO: 94ASGGGRMKQLEPKVEELLPKNYHLENEVARLNALVGERHHHHHH SEQ ID NO: 95ASGGGAMKQLEPKVEELLPKNYHLENEVARLAALVGERHHHHHH SEQ ID NO: 96ASGGGAMKQLEPKVEELLPKNYHLENEVARLQALVGERHHHHHH SEQ ID NO: 97ASGGGAMKQLEPKVEELLPKNYHLENEVARLNALVGERHHHHHH

It should be understood that A-X and Y-B fusions may be generated invarious orientations which means that the polynucleotide constructsencoding such fusion may be designed to express X or A in bothorientations (A-X where A's C-terminal is fused to X's N-terminal or X-Awhere X's C-terminal is fused to A's N-terminal). The same applies tothe Y-B fusion.

Irrespective of whether A, X, Y or B is at the N-terminal of the fusion,the polynucleotide sequence to generate such fusions will comprise anucleotide sequence designed to encode a signal peptide sequence, at thevery N-terminal of the fusion, for assisting extracellular release. Thesignal peptide is ultimately cleaved from the mature fusion. Preferredsignal peptide sequences are shown in Table 1A with SEQ ID NOs: 100-103.

In one embodiment (in particular where A is a Fab, Fab′) Y isindependently selected from, a Fab fragment, Fab′ fragment, scFv, asdAband a peptide, with the proviso that when X is a Fab fragment, aFab′ fragment, a scFv or sdAbthen Y is an antigen, such as a peptide,and when X is an antigen, such as a peptide, Y is a Fab fragment, a Fab′fragment, a scFv or a sdAb.

In one embodiment (in particular where A is a Fab, Fab′) Y isindependently selected from, a scFv, a sdAb, with the proviso that whenX is an antigen, such as a peptide.

In one embodiment (in particular where A is a Fab, Fab′) Y is a peptide,with the proviso that X is a scFv or sdAb.

In one embodiment (in particular where A is a scFv) Y is independentlyselected from, a Fab fragment, Fab′ fragment, scFv, a sdAband a peptide,with the proviso that when X is a Fab fragment, Fab′ fragment, scFv, asdAbthen Y is a peptide, and when X is a peptide Y is a Fab fragment, aFab′ fragment, a scFv or a sdAb.

In one embodiment (in particular where A is a scFv) Y is independentlyselected from, a, scFv, a sdAb and a peptide, with the proviso that whenX is a Fab fragment, Fab′ fragment, scFv, a sdAb then Y is a peptide,and when X is a peptide Y is a Fab fragment, a Fab′ fragment.

In one embodiment (in particular where A or B is a scFv) Y isindependently selected from, a Fab fragment, Fab′ fragment, with theproviso that X is a peptide

In one embodiment (in particular where A or B is a scFv) X isindependently selected from, a Fab fragment, Fab′ fragment, with theproviso that Y is a peptide

Thus the A and B elements of the bispecific format of the disclosuretogether independently represent:

-   -   a Fab or Fab′ arm and a scFv or sdAb arm, or    -   two scFvs arms, or,    -   two sdAbarms, or,    -   a scFv arm and a sdAbarm and

the X and Y components together independently represent:

-   -   a peptide and a Fab or Fab′ fragment, or    -   a peptide and a scFv, or    -   a peptide and a sdAb.

In one embodiment A-X is:

-   -   1. a Fab or Fab′ a linker and a peptide,    -   2. a Fab or Fab′ a linker and a scFv, or    -   3. a Fab or Fab′ a linker and a sdAb.

In one embodiment B-Y is:

-   -   4. a scFv a linker and a peptide,    -   5. a scFv a linker and a scFv, or    -   6. a scFv a linker and a sdAb.

In one embodiment the bispecific protein complex of the presentdisclosure is a combination, based on the numbers above, shown in Table1C:

TABLE 1C A-X B-Y 1 4 1 5 1 6 2 4 2 5 2 6 3 4 3 5 3 6

This type of arrangement is ideal for use in screening the units A-X andunit B-Y can be expressed.

Table 1D gives an overview of all possible combinations according to thescope of the present invention.

TABLE 1D A X Y B 1 Fab scFv peptide scFv 2 Fab scFv peptide sdAb 3 scFvscFv peptide scFv 4 scFv scFv peptide sdAb 5 sdAb scFv peptide sdAb 6Fab scFv antigen scFv 7 Fab scFv antigen sdAb 8 scFv scFv antigen scFv 9scFv scFv antigen sdAb 10 sdAb scFv antigen sdAb 11 Fab Fab peptide scFv12 Fab Fab peptide sdAb 13 scFv Fab peptide scFv 14 scFv Fab peptidesdAb 15 sdAb Fab peptide sdAb 16 Fab Fab antigen scFv 17 Fab Fab antigensdAb 18 scFv Fab antigen scFv 19 scFv Fab antigen sdAb 20 sdAb Fabantigen sdAb 21 Fab sdAb peptide scFv 22 Fab sdAb peptide sdAb 23 scFvsdAb peptide scFv 24 scFv sdAb peptide sdAb 25 sdAb sdAb peptide sdAb 26Fab sdAb antigen scFv 27 Fab sdAb antigen sdAb 28 scFv sdAb antigen scFv29 scFv sdAb antigen sdAb 30 sdAb sdAb antigen sdAb 31 Fab peptide scFvscFv 32 Fab peptide scFv sdAb 33 scFv peptide scFv scFv 34 scFv peptidescFv sdAb 35 sdAb peptide scFv sdAb 36 Fab antigen scFv scFv 37 Fabantigen scFv sdAb 38 scFv antigen scFv scFv 39 scFv antigen scFv sdAb 40sdAb antigen scFv sdAb 41 Fab peptide Fab scFv 42 Fab peptide Fab sdAb43 scFv peptide Fab scFv 44 scFv peptide Fab sdAb 45 sdAb peptide FabsdAb 46 Fab antigen Fab scFv 47 Fab antigen Fab sdAb 48 scFv antigen FabscFv 49 scFv antigen Fab sdAb 50 sdAb antigen Fab sdAb 51 Fab peptidesdAb scFv 52 Fab peptide sdAb sdAb 53 scFv peptide sdAb scFv 54 scFvpeptide sdAb sdAb 55 sdAb peptide sdAb sdAb 56 Fab antigen sdAb scFv 57Fab antigen sdAb sdAb 58 scFv antigen sdAb scFv 59 scFv antigen sdAbsdAb 60 sdAb antigen sdAb sdAb 61 scFv scFv peptide Fab 62 sdAb scFvpeptide Fab 63 sdAb scFv peptide scFv 64 scFv scFv antigen Fab 65 sdAbscFv antigen Fab 66 sdAb scFv antigen scFv 67 scFv Fab peptide Fab 68sdAb Fab peptide Fab 69 sdAb Fab peptide scFv 70 scFv Fab antigen Fab 71sdAb Fab antigen Fab 72 sdAb Fab antigen scFv 73 scFv sdAb peptide Fab74 sdAb sdAb peptide Fab 75 sdAb sdAb peptide scFv 76 scFv sdAb antigenFab 77 sdAb sdAb antigen Fab 78 sdAb sdAb antigen scFv 79 scFv peptidescFv Fab 80 sdAb peptide scFv Fab 81 sdAb peptide scFv scFv 82 scFvantigen scFv Fab 83 sdAb antigen scFv Fab 84 sdAb antigen scFv scFv 85scFv peptide Fab Fab 86 sdAb peptide Fab Fab 87 sdAb peptide Fab scFv 88scFv antigen Fab Fab 89 sdAb antigen Fab Fab 90 sdAb antigen Fab scFv 91scFv peptide sdAb Fab 92 sdAb peptide sdAb Fab 93 sdAb peptide sdAb scFv94 scFv antigen sdAb Fab 95 sdAb antigen sdAb Fab 96 sdAb antigen sdAbscFv

In one embodiment a scFv in A comprises an intravariable domaindisulfide bond.

In one embodiment a scFv in B comprises an intravariable domaindisulfide bond.

In one embodiment a scFv in X comprises an intravariable domaindisulfide bond.

In one embodiment a scFv in Y comprises an intravariable domaindisulfide bond.

In one embodiment the disulfide bond is between (unless the contextindicates otherwise Kabat numbering is employed in the list below).Wherever reference is made to Kabat numbering the relevant reference isKabat et al., 1987, in Sequences of Proteins of Immunological Interest,US Department of Health and Human Services, NIH, USA):

-   -   VH37+VL95C see for example Protein Science 6, 781-788 Zhu et al        (1997);    -   VH44+VL100 see for example; Biochemistry 33 5451-5459 Reiter et        al (1994); or Journal of Biological Chemistry Vol. 269 No. 28        pp. 18327-18331 Reiter et al (1994); or Protein Engineering,        vol. 10 no. 12 pp. 1453-1459 Rajagopal et al (1997);    -   VH44+VL105 see for example J Biochem. 118, 825-831 Luo et al        (1995);    -   VH45+VL87 see for example Protein Science 6, 781-788 Zhu et al        (1997);    -   VH55+VL101 see for example FEBS Letters 377 135-139 Young et al        (1995);    -   VH100+VL50 see for example Biochemistry 29 1362-1367 Glockshuber        et al (1990);    -   VH100b+VL49;    -   VH98+VL 46 see for example Protein Science 6, 781-788 Zhu et al        (1997);    -   VH101+VL46    -   VH105+VL43 see for example; Proc. Natl. Acad. Sci. USA Vol. 90        pp. 7538-7542 Brinkmann et al (1993); or Proteins 19, 35-47 Jung        et al (1994) or    -   VH106+VL57 see for example FEBS Letters 377 135-139 Young et al        (1995).

The amino acid pairs listed above are in the positions conducive toreplacement by cysteines such that disulfide bonds can be formed.Cysteines can be engineered into these positions by known techniques.

Accordingly in one embodiment a variable domain pair (VH/VL) of thepresent invention may be linked by a disulfide bond between two cysteineresidues, one in VH and one in VL, wherein the position of the pair ofcysteine residues is selected from the group consisting of VH37 andVL95, VH44 and VL100, VH44 and VL105, VH45 and VL87, VH100 and VL50,VH100b and VL49, VH98 and VL46, VH101 and VL46, VH105 and VL43 and VH106and VL57.

In one embodiment a variable domain pair (VH/VL) of the presentinvention may be linked by a disulfide bond between two cysteineresidues, one in VH and one in VL, which are outside of the CDRs whereinthe position of the pair of cysteine residues is selected from the groupconsisting of VH37 and VL95, VH44 and VL100, VH44 and VL105, VH45 andVL87, VH100 and VL50, VH98 and VL46, VH105 and VL43 and VH106 and VL57.

In one embodiment a variable domain pair (VH/VL) of the presentinvention may be linked by a disulfide bond between two cysteineresidues, one in VH and one in VL, which are outside of the CDRs whereinthe position of the pair of cysteine residues is selected from the groupconsisting of VH37 and VL95, VH44 and VL105, VH45 and VL87, VH100 andVL50, VH98 and VL46, VH105 and VL43 and VH106 and VL57.

In one embodiment a variable domain pair (VH/VL) of the presentinvention may be linked by a disulfide bond between two cysteineresidues wherein the cysteine residue of VH is at position 44 and thecysteine residue of VL is at position 100.

Typically the cysteine pairs are engineered into those positions in VHand VL, accordingly in one embodiment a variable domain pair (VH/VL) ofthe present invention may be linked by a disulfide bond between twoengineered cysteine

Thus, the amount of purification required after expression of each unit(A-X or B-Y) is minimal or in fact, unnecessary. The bispecific complexcan be formed in a 1:1 molar ratio by simply admixing the relevant unitsi.e. without recourse to conjugation and coupling chemistry. Wherepresent, the constant regions in the Fab/Fab′ fragment drivedimerization of the Fab/Fab′ component(s) and the binding partners X andY drive the equilibrium further in favour of forming the requisiteheterodimer bispecific complex. Again little or no purification isrequired after formation of the complex after heterodimerisation. Thuslarge number of A-X and B—Y can be readily prepared and combined.

Where one or both of A and B represent a scFv this may be advantageousbecause it allows scFv directly from a library to be used in the formatof the present disclosure, allowing rapid testing and avoiding the needto reformat the variable regions into an alternative format, such as aFab.

Obvious alternatives of the bispecific protein complex according to theinvention may be contemplated. One example include molecules whichcomprises more than one A or more than one B, such as a A′-A-X:Y-B orA-X:Y-B-B′, where A′ and B′ can each be independently selected from ascFv, a sdAb, a Fab or an antigen and is fused to A. For example, theA′-A part of the molecule may be formed by two scFv, each directed to adifferent epitope on the same target, forming the molecule(scFv)₂-X:Y-B.

In another example the bispecific complex may be formed by non-Ig-likebinding proteins, which include but are not imited to, adnectins,lipocalins, Kunitz domain-based binders, avimers, knottins, fynomers,atrimers, cytotoxic T-lymphocyte associated protein-4 (CTLA4)-basedbinders, darpins, affibodies, affilins, armadillo repeat proteins orcombinations thereof.

The bispecific protein complex according to the invention lacks an Fcfragment. The ability to prepare and screen a bispecific complex lackingthe Fc fragment CH2-CH3 also ensures that the biological activityobserved is in fact due solely to the variable region pairs in thecomplex. The simplicity of the bispecific complex of the disclosure andthe methods of preparing it are a huge advantage in the context offacilitating high-through-put screening of variable domain pairs to findnew target antigen combinations and also to optimise variable regionsequences for a given combination.

In one embodiment A and/or B is specific for an antigen selected fromthe group comprising: cell surface receptors such as T cell or B cellsignalling receptors, co-stimulatory molecules, checkpoint inhibitors,natural killer cell receptors, Immunolglobulin receptors,immunoglobulin-like receptors, matrix metalloproteases and membrane typematrix metalloproteases tissue inhibitors of metalloproteases, TNFRfamily receptors, B7 family receptors, adhesion molecules, integrins,cytokine/chemokine receptors, GPCRs, growth factor receptors, kinasereceptors, tissue-specific antigens, cancer antigens (tumour associatedantigens & peptides), pathogen recognition receptors, complementreceptors, hormone receptors, scavenger receptors, or soluble moleculessuch as cytokines, chemokines, leukotrienes, growth factors, hormones orenzymes or ion channels, including post translationally modified versionthereof, fragments thereof comprising at least one epitope.

In one embodiment there is provided a composition, for example apharmaceutical composition comprising one or more bispecific complexesaccording to the present disclosure.

Furthermore, the present inventors have devised a method of detectingsynergistic function in a heterodimerically-tethered bispecific proteincomplex of formula A-X:Y-B wherein:

-   -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   : is a binding interaction between X and Y;    -   A is a first protein component of the bispecific protein complex        independently selected from the group comprising a Fab fragment,        a Fab′ fragment, a sdAb and a single chain Fv (scFv);    -   B is a single chain Fv or sdAb;    -   X is a first binding partner of a binding pair independently        selected from an antigen, a Fab fragment, a Fab′ fragment, a        single chain Fv and sdAb; and    -   Y is a second binding partner of the binding pair independently        selected from antigen, a Fab fragment, a Fab′ fragment, a single        chain Fv and a sdAb;    -   with the proviso that when X is an antigen Y is a Fab fragment,        a Fab′ fragment, a single chain Fv or a sdAb specific to the        antigen represented by X and when Y is an antigen X is a Fab        fragment, a Fab′ fragment, a single chain Fv or a sdAbspecific        to the antigen represented by Y, said method comprising the        steps of:        -   (i) testing for activity in a functional assay for part or            all of a multiplex comprising at least one            heterodimerically-tethered bispecific protein complex; and        -   (ii) analysing the readout(s) from the functional assay to            detect synergistic biological function in the            heterodimerically-tethered bispecific protein complex.

Furthermore, the present inventors have devised a method of detectingsynergistic function in a heterodimerically-tethered bispecific proteincomplex of formula A-X:Y-B wherein:

-   -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   : is a binding interaction between X and Y;    -   A is a first protein component of the bispecific protein complex        independently selected from the group comprising a Fab fragment,        a Fab′ fragment, a sdAb and a single chain Fv (scFv);    -   B is a single chain Fv or sdAb;    -   X is a first binding partner of a binding pair independently        selected from an antigen, a Fab fragment, a Fab′ fragment, a        single chain Fv and sdAb; and    -   Y is a second binding partner of the binding pair independently        selected from antigen, a Fab fragment, a Fab′ fragment, a single        chain Fv and a sdAb;    -   with the proviso that when X is an antigen Y is a Fab fragment,        a Fab′ fragment, a single chain Fv or a sdAb specific to the        antigen represented by X and when Y is an antigen X is a Fab        fragment, a Fab′ fragment, a single chain Fv or a sdAb specific        to the antigen represented by Y, said method comprising the        steps of:        -   (i) testing for activity in a functional assay for part or            all of a multiplex comprising at least one            heterodimerically-tethered bispecific protein complex; and        -   (ii) analysing the readout(s) from the functional assay to            detect synergistic biological function in the            heterodimerically-tethered bispecific protein complex.

In one embodiment the multiplex is in the form of a grid, for examplethe multiplex comprises at least two heterodimerically-tetheredbispecific protein complexes.

Details provided above for the format apply equally to the formatemployed in the method of the present disclosure.

In one embodiment the heterodimerically tethered bispecific proteincomplexes are not purified prior to testing.

In one embodiment the A-X and Y-B fusion proteins are expressedtransiently and not purified before being mixed in a 1:1 molar ratio togenerate each heterodimerically tethered bispecific protein complex.

Thus generally the fusion proteins A-X and B—Y are not co-expressed inthe same cell. This is advantageous because it allows, for example 100fusion proteins to expressed and optionally purified and the subsequentmixing of the 100 fusion proteins in the various permutations canprovide 10,000 heterodimerically-tethered bispecific protein complexes,of which 5,000 are unique pairs.

In contrast certain prior art methods require co-expression ofbispecifics and thus for 10,000 complexes, 10,000 transfections,expressions and purifications are required.

However, if desired the A-X and B—Y may be expressed in the same cell.

The binding partners X and Y have affinity for each other and act asbiological equivalent of Velcro® or a bar and magnet and hold thecomplex together. Advantageously, this means that the fusion proteinsA-X and Y-B can be readily assembled into a bispecific protein complexsimply by mixing the fusion proteins together. Thus the bispecificprotein complex of the present disclosure has a modular structure whichallows for two different proteins to be easily assembled in order toproduce large panels of permutations of bispecific protein complexeswith different combinations of antigen specificities in, for example agrid-like fashion. This allows for the efficient and systematicscreening of a large number of bispecific protein complexes in order todetect additive, synergistic or novel biological function.

Given X and Y are specific for each other this significantly reduces theability to form homodimers. X and Y are collectively referred to hereinas a binding pair or binding partners. In one embodiment X does not havehigh affinity for other Xs. In one embodiment Y does not have highaffinity for other Ys. Advantageously, when X and Y do not formhomodimers, this prevents the formation of undesired monospecificprotein complexes, increases yield of the desired bispecific proteincomplexes, and removes the need for onerous purification steps to removethe monospecific protein complexes.

This allows rapid assembly of bispecific protein complexes with a yieldand/or purity which cannot be obtained efficiently by most prior artmethods, in particular prior art methods generally require extensivepurification steps. The yield of bispecific complex is typically 75% orhigher in the present invention.

Further advantageously, the bispecific protein complexes allow for thescreening of complexes wherein the constituent proteins (includingantigens bound by the constituent proteins) do not have a knownrelationship or are in different potentially unrelated pathways, suchas, two proteins which function in two distinct pathways and, forexample which the skilled person would not normally expect to come intocontact with each other can be tested in a bispecific protein complex toidentify additive, synergistic and/or novel function.

Furthermore multiple binding regions (such as variable regions) to agiven antigen or epitope can be investigated in parallel to identifynuances in biological function. This allows combinations of variableregion sequences directed to a given pair of antigens to be investigatedand optimised.

The present method allows the science to show the results and does notrely on pre-conceived ideas and technical prejudice about the biologicalfunction. This approach is potentially very powerful.

Advantageously the X and Y components allow a multiplex comprisingbispecific protein complexes made up of different permutations of fusionproteins to be assembled rapidly and easily.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing the structure and assembly of abispecific protein complex of the present disclosure.

FIG. 2 is a table showing an example 4×4 grid for functional screeningusing the bispecific antibody of the present invention. Using this grid,16 different bispecific protein complexes can be assembled andefficiently screened for synergistic function.

FIG. 3 is a cartoon of embodiments of the bispecific protein complexaccording to the invention where A and B are each independentlyrepresented by a Fab, a scFv or a sdAb; X, the anti-GCN4 peptide such asFab, scFv, sdAb and Y is the GCN4 peptide.

FIG. 4 is a cartoon of embodiments of the bispecific protein complexaccording to the invention where A is represented by a Fab; X, theanti-GCN4 peptide, is represented by a Fab, scFv, sdAb; Y is the GCN4peptide and B is represented by a Fab, a scFv or a sdAb.

FIG. 5 is a cartoon of embodiments of the bispecific protein complexaccording to the invention where A is represented by a scFv; X, theanti-GCN4 peptide, is represented by a Fab, scFv, sdAb; Y is the GCN4peptide and B is represented by a Fab, a scFv or a sdAb.

FIG. 6 is a cartoon of embodiments of the bispecific protein complexaccording to the invention where A is represented by a sdAb; X, theanti-GCN4 peptide, is represented by a Fab, scFv, sdAb; Y is the GCN4peptide and B is represented by a Fab, a scFv or a sdAb.

FIG. 7 Mammalian expression vector for scFv-Y format

FIG. 8 Inhibition of PLCg2 (+/−SD) by CD79-CD22 and CD79-CD45Fab-X:Fab-Y and Fab-X:scFv-Y bispecific combinations on IgM stimulatedB-cells from donor UCB Cone 130

FIG. 9 Inhibition of Akt (+/−SD) by CD79-CD22 and CD79-CD45 Fab-X:Fab-Yand Fab-X:scFv-Y bispecific combinations on IgM stimulated B-cells fromdonor UCB Cone 130

DETAILED DESCRIPTION

“Bispecific protein complex” as used herein refers to a moleculecomprising two proteins (A and B referred to herein as bispecificcomponents also referred to herein as the first protein component andsecond protein component, respectively of the bispecific) which areretained together by a heterodimeric-tether. Generally one or both ofthe proteins comprises a binding domain, preferably an antibody domainbut other binding domains could also be employed. When the bindingdomain comprises an antibody domain, each domain comprises at least 3complementarity determining regions (CDRs) and framework, for example aVHH comprises 3 CDRs whilst a Fab comprises 6 CDRs.

“Fusion proteins” as employed herein comprise a protein component A or Bfused to a binding partner X or Y (as appropriate). In one embodimentthe fusion protein is a translational protein expressed by recombinanttechniques from a genetic construct, for example expressed in a hostfrom a DNA construct. In the context of the present disclosure one ofthe key characteristics of a fusion protein is that it can be expressedas a “single protein/unit” from a cell (of course in the case of fusionproteins comprising a Fab/Fab′ fragment there will be two chains butthis will be considered a single protein for the purpose of the presentspecification with one chain, preferably the heavy chain fused at itsC-terminus to X or Y as appropriate, optionally via a linker asdescribed herein below; other orientations such as fusion to theN-terminus to X and Y are also possible).

The function of the heterodimeric tether X:Y is to retain the proteins Aand B in proximity to each other so that synergistic function of A and Bcan be effected or identified, for example employing the methoddescribed herein.

The term “heterodimeric-tether” as used herein refers to a tethercomprising two different binding partners X and Y which form aninteraction: (such as a binding) between each other which has an overallaffinity that is sufficient to retain the two binding partners together.In one embodiment X and/or Y are unsuitable for forming homodimers.

Heterodimerically-tethered and heterodimeric-tether are usedinterchangeably herein.

In one embodiment “unsuitable for forming homodimers” as employed hereinrefers to formation of the heterodimers of X-Y are more preferable, forexample more stable, such as thermodynamically stable, once formed thanhomodimers. In one embodiment the binding interaction between X and Y ismonovalent.

In one embodiment the X-Y interaction is more favourable than the X-X orY-Y interaction. This reduces the formation of homodimers X-X or Y-Ywhen the fusion proteins A-X and B—Y are mixed. Typically 75%heterodimer or more is formed following 1:1 molar ratio mixing.

If desired, a purification step (in particular a one-step purification),such as column chromatography may be employed, for example to purify thefusion protein units and/or bispecific protein complexes according tothe present disclosure.

In one embodiment a purification step is provided after expression ofthe or each fusion protein, although typically aggregate levels are low.Thus in one embodiment prior to in vitro mixing, the fusion protein(s)is/are provided in substantially pure form. Substantially pure form asemployed herein refers to wherein the fusion protein is 90, 91, 92, 93,94, 95, 96, 97, 98, 99 or 100% monomer.

In one embodiment no purification of the fusion protein or proteins isperformed.

In one embodiment each fusion protein unit is expressed in a differentexpression experiment/run.

In one embodiment no purification of the fusion protein or proteins isperformed before mixing to generate a bispecific protein complex. In oneembodiment no purification of the fusion protein or proteins isperformed before and/or after mixing.

In one embodiment no purification is required after the bispecificprotein complex formation.

In one embodiment after mixing, and generally without furtherpurification, at least 50% of the composition is the desired bispecificprotein complex, for example at least 60, 65, 70, 75, 80% of thecomposition is the required bispecific protein complex.

In one embodiment the ratio of fusion proteins employed in the in vitromixing step of the present method is A-X to B-Y 0.8:1 to 3:1, such as1.5:1 or 2:1.

In one embodiment the ratio of fusion proteins employed in the in vitromixing step of the present method is B-Y to A-X 0.8:1 to 3:1, such as1.5:1 or 2:1, in a particular a molar ratio. In one embodiment the ratioof A-X to B-Y employed in the in vitro mixing step is 1:1, in particulara 1:1 molar ratio.

The present disclosure also extends to a method of preparing abispecific complex according to the present disclosure comprisingadmixing a fusion protein A-X and B-Y, for example in a 1:1 molar ratio.

In one embodiment the mixing occurs in vitro.

In one embodiment mixing occurs in a cell, for example a host cell.

In one embodiment, the mixing occurs in vivo, i.e. the fusion proteinsA-X and B-Y interact with each other within a subject's body to form theheterodimeric-tether and in consequence, the bispecific protein complex.

In one embodiment, X and Y are completely specific for each other and donot bind to any other peptides/proteins in a cell or within a subject'sbody. This can be achieved for example by ensuring that X and Y are notnaturally present in the target cell or in the target subject's body.This can be achieved, for example by selecting X or Y to be from aspecies or entity which is different to the subject (e.g. a yeastprotein) and ensuring the other variable is specific to it.Advantageously, this prevents the binding of the fusion proteins A-Xand/or B-Y to an undesired target, thereby generating unwantedoff-target effects.

In one embodiment one (or at least one) of the binding partners isincapable of forming a homodimer, for example an amino acid sequence ofthe binding partner is mutated to eliminate or minimise the formation ofhomodimers.

In one embodiment both of the binding partners are incapable of forminga homodimer, for example an amino acid sequence of the peptide bindingpartner is mutated to eliminate or minimise the formation of homodimersand a sdAb specific thereto is employed.

Incapable of forming homodimers or aggregates as employed herein, refersto a low or zero propensity to form homodimers or aggregate. Low asemployed herein refers to 5% or less, such as 4, 3, 2, 1, 0.5% or lessaggregate, for example after mixing or expression or purification.

Small amounts of aggregate in the fusion proteins or residual in theheterodimerically-tethered bispecific protein complex generally hasminimal effect on the screening method of the present disclosure.Therefore, in one embodiment no purification of fusion protein(s) and/orbispecific protein complex(es) is/are employed in the method, inparticular after the mixing step.

In one embodiment: is a binding interaction based on attractive forces,for example Van der Waals forces, such as hydrogen bonding andelectrostatic interactions, in particular, based on antibody specificityfor an antigen (such as a peptide).

In one embodiment: is a covalent bond formed from a specific chemicalinteraction, such as click chemistry. In one embodiment: is not acovalent bond. In one embodiment conjugation/coupling chemistry is notemployed to prepare the bispecific protein complexes of the presentdisclosure.

“Form the complex” as employed herein refers to an interaction,including a binding interaction or a chemical reaction, which issufficiently specific and strong when the fusion protein components A-Xand B—Y are brought into contact under appropriate conditions that thecomplex is assembled and the fusion proteins are retained together.

“Retained together” as employed herein refers to the holding of thecomponents (the fusion proteins) in the proximity of each other, suchthat after X:Y binding the complex can be handled as if it were onemolecule, and in many instances behaves and acts like a single molecule.In one embodiment the retention renders the complex suitable for use inthe method disclosed herein, i.e. suitable for use in at least onefunctional screen.

Specificity as employed herein refers to where, for example the partnersin the interaction e.g. X:Y or A and antigen or B and antigen onlyrecognise each other or have significantly higher affinity for eachother in comparison to non-partners, for example at least 2, 3, 4, 5, 6,7, 8, 9, 10 times higher affinity, than for example a background levelof binding to an unrelated non partner protein.

Specificity in relation to X and Y as employed herein refers to wherethe binding partners X and Y in the interaction only recognise eachother or have significantly higher affinity for each other in comparisonto non-partners, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10 timeshigher affinity.

In one embodiment the binding interaction is reversible. In oneembodiment the binding interaction is essentially irreversible.

Essentially irreversible as employed herein refers to a slow off rate(dissociation constant) of the antibody or binding fragment.

In one embodiment, the binding interaction between X and Y has a lowdissociation constant. Examples of a low dissociation constant include1-9×10⁻² s⁻¹ or less, for example 1-9×10⁻³ s⁻¹, 1-9×10's⁻¹, 1-9×10⁻⁵s⁻¹, 1-9×10 s⁻¹ or 1-9×10 s⁻¹. Particularly suitable dissociationconstants include 2×10⁻⁴ s⁻¹ or less, for example 1×10⁻⁵ s⁻¹, 1×10⁻⁶ s⁻¹or 1×10⁻⁷ s⁻¹.

Whilst not wishing to be bound by theory it is thought that the lowdissociation constant (also referred to as off rate) allows themolecules to be sufficiently stable to render the bispecific proteincomplex useful, in particular in functional screening assays.

In one embodiment, the affinity of X and Y for each other is 5 nM orstronger, for example 900 pM or stronger, such as 800, 700, 600, 500,400 or 300 pM.

Affinity is a value calculated from the on and off rate of the entity.The term “affinity” as used herein refers to the strength of the sumtotal of non-covalent interactions between a single binding site of amolecule (e.g. an antibody) and its binding partner (e.g. a peptide).The affinity of a molecule for its binding partner can generally berepresented by the dissociation constant (KD). Affinity can be measuredby common methods known in the art, including those described herein,such as surface plasmon resonance methods, in particular BIAcore.

However, the ability to hold the complex together is not just aboutaffinity. Whilst not wishing to be bound by theory, we hypothesise thatin fact there are three significant components: the on-rate, off-rateand the affinity. The calculation for affinity is based on on-rate andoff-rate. So if the on-rate is low and the off-rate is fast, then theaffinity will be low and that will not be sufficient to hold thebispecific protein complex together. However, a slow on-rate could becompensated for by a slow off-rate giving an overall suitable affinity.In some embodiments a high on-rate may be sufficient to hold the complextogether.

If the binding partners (X and Y) employed in the complex have a slowon-rate then additional time may be required after mixing the componentsto allow the complex to form. If the affinity between the bindingpartners is sufficiently high, it may be possible for the bispecificprotein complex to perform its desired biological function even if theaffinity of the proteins (A and B) of the bispecific protein complexonly bind weakly to their targets. Conversely, if the proteins (A and B)are able to bind strongly to their targets, it may be possible toachieve the same biological function even if the affinity of the bindingpartners (X and Y) for each other is lower. In other words, a ‘trinity’relationship exists such that a higher affinity between the bindingpartners can compensate for a lower affinity for the targets and viceversa.

In one embodiment the method herein is employed to screen a phagedisplay library, including a naïve phage library, by preparing fusionproteins of the disclosure from the library.

The bispecific protein complexes of the present invention may be used inany suitable application, including functional screening. This novelformat is particularly useful in multiplex functional screening toidentify protein targets based on function, and optimal epitopes onthose target proteins, which could be targeted by bispecific therapies.Furthermore where proteins A and B are antibodies or binding fragmentsthereof the bispecific protein complexes may also be used for multiplexfunctional screening to identify optimal variable region pairs for usein bispecific antibody therapeutics.

“Multiplex” as employed herein is a population of entities for testingcomprising:

-   -   at least two component fusion proteins (A-X and Y-B) combined to        generate at least one heterodimerically-tethered bispecific        protein complex and at least one relevant biological comparator        in the same or a different format, or    -   at least two heterodimerically-tethered bispecific protein        complexes with optionally at least one relevant biological        comparator in the same or a different format.

Clearly to be useful, the different format employed as the comparatormust be suitable for testing in a functional in vitro assay employed inthe disclosure. In one example the comparator in the multiplex is amonovalent mixture of A-X and B-X or a bivalent monospecific complex ofA-X-Y-A.

In one embodiment the multiplex comprises 1 to hundreds of thousands ofheterodimerically-tethered bispecific protein complexes, for example 2to 500,000 of said complexes, such as 2 to 100,000 or 2 to 10,000, inparticular generated from mixing in a grid 2 to 100s of first and secondfusion proteins (A-X and B-Y). In one embodiment the multiplex comprisesfor example 2 to 1,000, such as 2 to 900, 2 to 800, 2 to 700, 2 to 600,2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 2 to 90, 3 to 80, 4 to70, 5 to 60, 6 to 50, 7 to 40, 8 to 30, 9 to 25, 10 to 20 or 15bispecific protein complexes. See FIG. 2 for an example of such a grid.

In one embodiment the number of heterodimerically-tethered bispecificproteins in this multiplex is n² where n is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more.

The multiplex may be in the form of an array, for example a microtitreplate, wherein each well of the microplate may contain a differentbispecific protein complex. The bispecific protein complexes may betethered to a solid substrate surface, for example attached to a bead,or they may be suspended in a liquid (e.g. a solution or media) form,for example within a well or within a droplet.

In one embodiment every ‘A’ in the multiplex is a different protein,preferably an antibody or binding fragment thereof that binds to atarget antigen and every ‘B’ is a different protein preferably anantibody or binding fragment thereof that binds to a target antigen.

In one embodiment the multiplex is provided in a grid as discussedbelow, for example an 8×8, 16×16 or 16×20, which equates to 64, 256 or320 samples respectively.

“Grid” as employed herein refers to a two dimensional plot or arraywhere one variable, such a protein A (in A-X) is varied along one axis,such as the X-axis (horizontal axis) and another variable such asprotein B (in B-Y) is varied along the other axis, such as the Y axis(vertical axis). This arrangement assists in systematically evaluatingthe various combinations (permutations) of the variables.

In one embodiment the multiplex is provided on 96 well plates and thesamples analysed may be multiples thereof i.e. 96, 192, 384 etc.

Advantageously, a grid arrangement is particularly advantageous forefficiently screening the biological function of bispecific proteincomplexes according to the present disclosure. FIG. 2 shows an exampleof such a grid, whereby 4 first fusion proteins can be readily combinedwith 4 second fusion proteins to produce 16 bispecific proteincomplexes.

Other variations of a screening grid will be apparent to the skilledaddressee, for example the first protein (A) in the first fusion protein(A-X) may be kept constant whilst the second protein (B) in the secondfusion protein (B-Y) is varied. This may be useful for quickly screeninga large number of different second proteins for synergistic functionwith the pre-selected first protein.

In another embodiment, protein A is varied along one axis by changingthe antibody variable regions of protein A such that each antibodyvariant is specific for the same antigen but has a different combinationof variable regions. Protein B may either be kept constant or may alsobe varied in the same fashion or varied such that the antigenspecificity changes (across or down the grid) for the B proteins.

Advantageously, such a screening grid may potentially allow for thedetection of small differences in synergistic function when thebispecific protein complexes are specific for the same antigens but withdifferent combinations of variable regions.

In one embodiment, a “common” first fusion protein (A-X) according tothe present disclosure may be present within each well. A range ofdifferent second fusion proteins (B-Y) according to the presentdisclosure may then be dispensed into each well. Subsequently, thespecific binding interaction of the two binding partners (X and Y)physically brings the two fusion proteins together to form thebispecific protein complexes. This results in a multiplex comprisingbispecific protein complexes which all bind to a common first targetantigen (bound by A) but are also capable of binding to a second targetantigen (bound by B) which may be different for each bispecific proteincomplex.

In one embodiment the B-Y fusion proteins comprise different variableregions to the same target antigen to allow optimisation of the variableregions and/or epitopes of the given target antigen bound by B whencombined with the variable regions in A-X.

“Common” first fusion protein as employed herein refers to fusionsproteins wherein the A or B component thereof, bind the same proteins orepitope, in particular where the A or B component have complete identityin the common fusion protein i.e. the common first fusion protein alwayscomprises the same variable region sequence.

The skilled person is also aware of different variations of the above,such that the desired specificities of the bispecific protein complexesat each position in the multiplex can be readily controlled. This allowsfor the efficient screening of different combinations of bispecificprotein complexes when such multiplexes are used in functional assays.In one embodiment factorial design is employed to define the variablesemployed in the grid.

In one embodiment the method of the present disclosure is conducive tohigh-throughput analysis.

In one embodiment, multiple bispecific protein complexes are tested inparallel or essentially simultaneously.

Simultaneously as employed herein refers to the where thesamples/molecules/complexes are analysed in the same analysis, forexample in the same “run”. This may be advantageous as generally thereagents employed for a given sample run will be the same batch,concentration, cell source etc and therefore have the same properties.Furthermore the environmental conditions under which the analysis isperformed, such as temperature and humidity are likely to be similar.

In one embodiment simultaneously refers to concomitant analysis wherethe signal output is analysed by an instrument at essentially the sametime. This signal may require deconvolution to interpret the resultsobtained.

Advantageously, testing multiple bispecific protein complexes allows formore efficient screening of a large number of bispecific proteincomplexes and the identification of new and interesting relationships.

In one embodiment, the multiple bispecific protein complexes are testedby using a multiplex as defined above and subjecting the same to one ormore functional assays. Accordingly the present invention provides amethod for detecting synergistic biological function in aheterodimerically-tethered bispecific protein complex of formula A-X:Y-B

-   -   wherein X:Y is a heterodimeric-tether    -   : is a binding interaction between X and Y,    -   A and B are protein components of the bispecific in the form of        fusion proteins with X and Y respectively, said method        comprising the steps of:        -   (i) testing for activity in a functional assay for part or            all of a multiplex comprising at least one            heterodimerically-tethered bispecific protein complex; and        -   (ii) analysing the readout(s) from the functional assay to            identify or detect synergistic biological function in the            heterodimerically-tethered bispecific protein complex; and    -   wherein Y is an antigen and X is an antibody or binding fragment        thereof specific to Y or X is an antigen and Y is an antibody or        binding fragment thereof specific to X.

The term “biological function” as used herein refers to an activity thatis natural to or the purpose of, the biological entity being tested, forexample a natural activity of a cell, protein or similar. Ideally thepresence of the biological function can be tested using an in vitrofunctional assay, including assays employing mammalian cells, such asliving cells, such as B or T cells, or tissue ex vivo. Natural functionas employed herein also includes aberrant function, such as functionsassociated with diseases, such as cancers.

A relevant “biological comparator” as employed herein refers to asuitable entity for assessing activity, in the same assay as thatemployed for the bispecific protein complex, to establish if there isany change or novel activity or function. Suitable comparators forA-X:Y-B may include a purified protein (including recombinant proteins)in a natural form or presented in the same format as the bispecific e.g.where A and B are the same entity, such as A-X:Y-A or B-X:Y-B i.e. abivalent monospecific complex. Alternatively the fusion protein A-X orB-Y in an uncomplexed form may be employed as a comparator alone or asan uncomplexed mixture such as A-X and B-X together or A-Y and B-Ytogether. Alternatively, multiple comparators of different formats (inparticular as described herein) may be employed. The person skilled inthe art is able to identify and include a suitable control/comparatorbased on common general knowledge or information that is found in theliterature.

The term “synergistic function” or “synergistic biological function” asused herein refers to a biological activity or level of biologicalactivity or an effect on a biological function or activity that:

-   -   is not observed with individual fusion protein components until        a bispecific is employed (and may include activity observed with        a combination of antibodies to the said antigens, which are not        in an bispecific format, but in particular refers to activity        only observed when the two binding domains are linked in a        bispecific format) or    -   higher or lower activity in comparison to the activity observed        when the first and second proteins of a bispecific protein        complex of the present disclosure are employed individually, for        example activity which is only observed in a bispecific form.

Therefore, “synergistic” includes novel biological function or novelactivity. Synergistic function as employed herein does not generallyinclude simple targeting i.e. based only on binding but will generallyinvolve some inhibition, activation, signalling or similar afterbinding.

Novel biological function or novel activity as employed herein refers toa biological function or activity which is not apparent or is absentuntil the two or more synergistic entities (protein A and protein B) arebrought together (as a bispecific or otherwise) or a previouslyunidentified function.

Higher as employed herein refers to an increase in activity including anincrease from zero e.g. some activity in the bispecific where theindividual uncomplexed bispecific component or components has/have noactivity in the relevant functional assay, also referred to herein asnew activity or novel biological function. Higher as employed hereinalso includes a greater than additive function in the bispecific in arelevant functional assay in comparison to the individual uncomplexedbispecific components (tested alone or in combination with beinglinked), for example 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300%or more increase in a relevant activity.

In one embodiment the uncomplexed proteins together have the sameactivity as the bispecific and this activity or function was previouslyunknown. This is also a novel synergistic function in the context of thepresent specification.

In one embodiment the synergistic function is a higher function.

In one embodiment the synergistic function is a lower function.

Lower function as employed herein refers to where the bispecific in therelevant functional assay has less or no activity in comparison to theindividual uncomplexed bispecific component (s) which has/have activityin the relevant functional assay, also referred to herein as newactivity or novel biological function (such as a natural protein i.e. arecombinant isolated protein which is not in a fusion protein nor partof any other complex other than one in which occurs in vivo-including anactive domain or fragment of said protein) analysed as an individualprotein or analysed as a mixture of proteins under the same conditions,for example 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300% or moredecrease in a relevant activity. Greater than 100% decrease in activityrefers to a gain in positive activity in a different direction, forexample where an entity is an agonist decrease in activity over 100% mayrender the entity an antagonist and vice versa.

In one embodiment the activity of the bispecific complex is lower thanthe sum of the known function of protein A and protein B.

In some embodiments the bispecific protein complexes of the presentdisclosure have simply additive biological function. Additive biologicalfunction as employed herein refers to function, which is the same as thesum of each of the components A and B individually, when tested underthe same conditions. An additive function may be a novel function if theactivity or function was previously unknown or unidentified.

Screening is performed using any suitable assay known in the art,depending on the desired function to be identified.

In one embodiment, the functional assay employed in a method of thepresent disclosure is an in vitro or ex vivO assay,

A “functional assay,” as used herein, is an assay that can be used todetermine one or more desired properties or activities of the bispecificprotein complexes, antibody complexes or the mixture of antibodiessubject to the assay conditions. Suitable functional assays may bebinding assays, apoptosis assays, antibody-dependent cellularcytotoxicity (ADCC) assays, complement-dependent cytotoxicity (CDC)assays, inhibition of cell growth or proliferation (cytostatic effect)assays, cell-killing (cytotoxic effect) assays, cell-signalling assays,cytokine production assays, antibody production and isotype switching,cellular differentiation assays, colony forming assays, chemotaxisassays, cell adhesion assays, cell migration assays, cell cycle assays,metabolic assays (whole cell and organelle function), assays formeasuring inhibition of binding of pathogen to target cell, assays tomeasure the secretion of vascular endothelial growth factor (VEGF) orother secreted molecules, assays for bacteriostasis, bactericidalactivity, neutralization of viruses, assays to measure the attraction ofcomponents of the immune system to the site where antibodies are bound,including in situ hybridization methods, labeling methods, and the like.

In one embodiment in vivo assays, such as animal models, including mousetumor models, models of auto-immune disease, virus-infected orbacteria-infected rodent or primate models, and the like, may beemployed.

The skilled person is well able to select a suitable functional assaybased on the target/proteins being investigated. However, the complexesmay be subject to a panel of “standard” assays without preselectingassays thought to be relevant in an attempt identify new functionality.

In the context of bispecific antibody complexes, the efficacy ofbispecific antibody complexes according to the present disclosure can becompared to individual antibodies or mixtures of antibodies (orfragments) in such models by methods generally known to one of ordinaryskill in the art.

For example, the bispecific antibody complexes may be tested for theability to inhibit proliferation, affect viability or metabolic activityof cells (for example with a stain such as allamar blue or by monitoringluminescence due to luciferase expressed by the cells), or causeapoptosis of cancer cells, which are biological functions that includeproperties other than binding to an antigen.

By choosing functional assays closely related to a particular disease ofinterest, the methods of the disclosure make it possible to identifypotentially therapeutic antibodies that bind to known or unknown targetmolecules. It is thus possible to identify new target molecules and/orto directly identify potentially therapeutic antibodies using themethods of the disclosure. Advantageously, the present method is notlimited to any particular assay(s) and provides the user with completeflexibility to select the most appropriate functional assay depending onthe requirements.

When screening the bispecific antibody complexes for desired biologicalfunction, various strategies may be employed. For example, mediumcontaining the antibodies can be directly screened for the biologicalactivity. Alternatively, the antibodies can be bound to beads coated orto microtiter plates prior to screening for biological activity.Alternatively a fusion protein maybe purified via a His tag in a nickelcapture purification step. Such strategies may increase localconcentrations of the antibodies leading to clearer results from thefunctional assays.

The functional assays may be repeated a number of times as necessarywith or without different samples of a particular bispecific antibodycomplex to enhance the reliability of the results. Various statisticaltests known to the skilled person can be employed to identifystatistically significant results and thus identify bispecific antibodycomplexes with biological functions.

When establishing a functional assay for screening the skilled personcan set a suitable threshold over which an identified activity is deemeda ‘hit’. Where more than one functional assay is used the threshold foreach assay may be set at a suitable level to establish a manageable hitrate. In one example the hit rate may be 3-5%. In one example thecriteria set when searching for pairs of antigens that inhibit B cellfunction may be at least 30% inhibition of at least two phospho-readoutsin a B cell activation assay.

In the bispecific protein complexes of the present invention thefollowing protein and peptide components may be used.

In one embodiment, at least one of the first binding partner, X, and thesecond binding partner, Y, of the binding pair are independentlyselected from a peptide and a protein; for example the first bindingpartner or second binding partner is a peptide.

Suitable peptides include the group comprising GCN4, Fos/Jun (human andmurine Fos have a Uniprot number P01100 and P01101 respectively andhuman and murine jun have a Uniprot number 05412 and 05627respectively), HA-tag which correspond to amino acids 98 to 106 of humaninfluenza hemagglutinin, polyhistidine (His), c-myc and FLAG. Otherpeptides are also contemplated as suitable for use in the presentdisclosure and particularly suitable peptides are affinity tags forprotein purification because such peptides have a tendency to bind withhigh affinity to their respective binding partners.

In one embodiment the peptide is not E5B9.

The term “peptide” as used herein refers to a short polymer of aminoacids linked by peptide bonds, wherein the peptide contains in the rangeof 2 to 100 amino acids, for example 5 to 99, such as 6 to 98, 7 to 97,8 to 96 or 5 to 25. In one embodiment a peptide employed in the presentdisclosure is an amino acid sequence of 50 amino acid residues or less,for example 40, 30, 20, 10 or less.

In one embodiment, the protein is an antibody or an antibody fragment.

The term “antibody” as used herein refers to an immunoglobulin moleculecapable of specific binding to a target antigen, such as a carbohydrate,polynucleotide, lipid, polypeptide, peptide, protein etc., via at leastone antigen recognition site (also referred to as a binding siteherein), located in the variable region of the immunoglobulin molecule.

As used herein the term “antibody” or “antibody molecule” includesantibodies and antigen-binding fragments thereof.

The term “antigen-binding fragment” of an antibody or “antibodyfragments” as employed herein refers to fragments of an antibody,naturally occurring or man-made, and includes but is not limited to Fab,modified Fab, Fab′, modified Fab′, F(ab′)2, Fv, single domain antibodies(sdAb), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies,triabodies, tetrabodies and epitope-binding fragments of any of theabove (see for example Holliger and Hudson, 2005, Nature Biotech.23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews—Online2(3), 209-217).

The methods for creating and manufacturing these antibody fragments arewell known in the art (see for example Verma et al., 1998, Journal ofImmunological Methods, 216:165-181). Other antibody fragments for use inthe present disclosure include the Fab and Fab′ fragments described inInternational patent applications WO05/003169, WO05/003170 andWO05/003171. Multi-valent antibodies may comprise multiple specificitiese.g. bispecific or may be monospecific (see for example WO92/22853,WO05/113605, WO2009/040562 and WO2010/035012).

An “antigen-binding fragment” as employed herein refers to a fragment,for example of an antibody or of another molecule, capable of binding atarget peptide or antigen with sufficient affinity to characterise thefragment as specific for the peptide or antigen.

The term “Fab fragment” as used herein refers to an antibody fragmentcomprising a light chain fragment comprising a VL (variable light)domain and a constant domain of a light chain (CL), and a VH (variableheavy) domain and a first constant domain (CH1) of a heavy chain. In oneexample the heavy chain sequences of the Fab fragment “terminates” atthe interchain cysteine of CH1. In one embodiment the Fab fragmentemployed in a fusion protein of the present disclosure, such as A-Xand/or B—Y is monovalent.

A Fab′ fragment as employed herein refers to a Fab fragment furthercomprising all or part of a hinge region. In one embodiment the Fab′fragment employed in a fusion protein of the present disclosure, such asA-X and/or B—Y is monovalent.

The term “single-chain Fv” or abbreviated as “scFv”, as used hereinrefers to an antibody fragment that comprises VH and VL antibody domainslinked (for example by a peptide linker) to form a single polypeptidechain. The constant regions of the heavy and light chain are omitted inthis format. Single-chain Fv as employed herein includes disulfidestabilised versions thereof wherein in addition to the peptide linker adisulfide bond is present between the variable regions.

Disulfide stabilised scFv may eliminate the propensity of some variableregions to dynamically breath, which relates to variable regionsseparating and coming together again. The term “sdAb” or “single domainantibodie(s)” as used herein refers to molecules comprising a singleantigen-binding domain. They may be artificially created or naturallyoccurring and include, but are not limited to, VH only, VL only, camelidVHHs, human domain antibodies, shark derived antibodies such as IgNARsand other non-antibody single domain binding formats, including but notlimited to, adnectins, lipocalins, Kunitz domain-based binders, avimers,knottins, fynomers, atrimers, cytotoxic T-lymphocyte associatedprotein-4 (CTLA4)-based binders, darpins, affibodies, affilins,armadillo repeat proteins. The antibody binding fragment and/or thebispecific antibody complex does not comprise an Fc region. “Does notcomprise an Fc region” as employed herein refers to the lower constantdomains, such as CH2, CH3 and CH4 which are absent. However, constantdomains such as CH1, CKappa/CLambda may be present.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is a monospecific antibody or antibodyfragment, in particular a monovalent Fab, Fab′, scFv, Fv, sdAb orsimilar.

In one embodiment, the antibody or antibody fragment employed in thesecond fusion protein (B-Y) is a monospecific antibody or antibodyfragment, in particular a monovalent Fab, Fab′, scFv or similar.

“Monospecific” as employed herein refers to the ability to bind only onetarget antigen.

“Monovalent” as employed herein refers to the antibody or antibodyfragment having a single binding site and therefore only binding thetarget antigen only once.

“Multivalent” as used herein refers to antibodies or fragments thereofhaving at least two binding sites capable of binding to two or moreepitopes with the same, identical specificity, e.g. repeating identicalunits on the surface of a virus particle.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is multivalent, that is has two or morebinding domains.

In one embodiment, the antibody or antibody fragment employed in thesecond fusion protein (B-Y) is multivalent, that is has two or morebinding domains.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is monovalent and the antibody or antibodyfragment employed in the second fusion protein (B-X) is monovalent.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is monovalent and the antibody or antibodyfragment employed in the second fusion protein (B-Y) is multivalent.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is multivalent and the antibody or antibodyfragment employed in the second fusion protein (B-Y) is monovalent.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is multivalent and the antibody or antibodyfragment employed in the second fusion protein (B-Y) is multivalent.

In one embodiment A-X or B—Y is not a fusion protein comprising twoscFvs one specific to the antigen CD33 and one specific to the antigenCD3 or alternatively a bispecific complex format specific to these twoantigens.

In one embodiment the A-X or B—Y is not a fusion protein comprising ascFv (or alternatively another antibody format) specific to CD3 linkedto a peptide E5B9.

A “binding domain or site” as employed herein is the part of theantibody that contacts the antigen/epitope and participates in a bindinginteraction therewith. In one embodiment the binding domain contains atleast one variable domain or a derivative thereof, for example a pair ofvariable domains or derivatives thereof, such as a cognate pair ofvariable domains or a derivative thereof.

In one embodiment a variable domain comprises 3 CDRs, in particular anantibody domain such as a VH, VL or sdAb. In one embodiment the bindingdomain comprises two variable domains and 6 CDRs and a framework andtogether these elements contribute to the specificity of the bindinginteraction of the antibody or binding fragment with theantigen/epitope.

A “cognate pair” as employed herein refers to a heavy and light chainpair isolated from a host as a pre-formed couple. This definition doesnot include variable domains isolated from a library, wherein theoriginal pairings from a host is not retained. Cognate pairs may beadvantageous because they are often affinity matured in the host andtherefore may have high affinity for the antigen to which they arespecific.

A “derivative of a naturally occurring domain” as employed herein isintended to refer to where one, two, three, four, five or more than fiveamino acids in a naturally occurring sequence have been replaced ordeleted, for example to optimize the properties of the domain such as byeliminating undesirable properties but wherein the characterizingfeature(s) of the domain is/are retained. Examples of modifications arethose to remove glycosylation sites or solvent exposed lysines. Thesemodifications can be achieved by replacing the relevant amino acidresidues with a conservative amino acid substitution.

In one embodiment, the bispecific antibody complexes of the presentdisclosure or antibody/fragment components thereof are processed toprovide improved affinity for a target antigen or antigens. Suchvariants can be obtained by a number of affinity maturation protocolsincluding mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403,1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783,1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol.,250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin.Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol.Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391,288-291, 1998). Vaughan et al. (supra) discusses these methods ofaffinity maturation.

In one embodiment, the first antibody or antibody fragment (A) isspecific to a first antigen and the second antibody or antibody fragment(B) is specific to a second antigen, wherein the first and secondantigens are different. Advantageously, the bispecfic antibody complexmay be specific for two different antigens. This presents thepossibility of the antibody complex binding to two different antigens,each located on a different entity, thereby bringing the two entitiesinto close physical proximity with each other.

Alternatively, the first antibody or antibody fragment (A) may bespecific for a first epitope and the second antibody or antibodyfragment (B) may be specific for a second epitope, wherein the first andsecond epitopes are both on the same antigen. This can greatly enhancethe avidity of the bispecific antibody complex for the antigen due tothe multiple interactions between the antigen and bispecific antibodycomplex.

In one embodiment, the first (A) or second (B) antibody fragment isselected from the group consisting of: a fragment antigen binding (Fab),a Fab′, a single chain variable fragment (scFv) and a single domainantibody (sdAb), such as a VHH.

For convenience bispecific protein complexes of the present disclosureare referred to herein as A-X:Y-B. However, this nomenclature is notintended to limit how the fusion protein A-X and B—Y are designedbecause our experiments indicate that binding partners X and Y can bereversed i.e. A-Y and B-X without adversely impacting on the method.Thus A and B and X and Y are nominal labels referred to for assistingthe explanation of the present technology. “Attached” as employed hereinrefers to connected or joined directly or indirectly for example via alinker, such as a peptide linker examples of which are discussed below.Directly connected includes fused together (for example a peptide bond)or conjugated chemically.

“Binding partner” as employed herein refers to one component part of abinding pair. In one embodiment, the affinity of the binding partners ishigh, 5 nM or stronger, such as 900, 800, 700, 600, 500, 400, 300 pM orstronger.

“Binding pair” as employed herein refers to two binding partners whichspecifically bind to each other. Examples of a binding pair include apeptide and an antibody or binding fragment specific thereto, or anenzyme and ligand, or an enzyme and an inhibitor of that enzyme.

In one embodiment X is attached via a linker (such as ASGGGG SEQ ID NO:71 or ASGGGGSG SEQ ID NO: 72 or ASGGG SEQ ID NO: 73 or AAASGGG SEQ IDNO: 74) or any other suitable linker known in the art or describedherein below, to the C-terminal of the heavy chain of the antibody orfragment (protein A) and Y is attached via a linker (such as ASGGGG SEQID NO: 71 or ASGGGGSG SEQ ID NO: 72 or ASGGG SEQ ID NO: 73 or AAASGGGSEQ ID NO: 74) to the C-terminal of the heavy chain of the antibody orfragment (protein B).

Examples of a suitable binding pair (X or Y) may include GCN4 (SEQ IDNO: 1 or lacking the HIS tag, amino acids 1-38 of SEQ ID NO: 1), avariant, a derivative or fragment thereof (for example any of thesequences shown by SEQ ID NOs: 75-97) and 52SR4 (SEQ ID NO: 3 or lackingthe HIS tag amino acids 1 to 243 of SEQ ID NO:3) or a variant thereof,which is a scFv specific for GCN4.

In a one embodiment, the first binding partner (nominally X) is GCN4(for example as shown in SEQ ID NO: 1) or a fragment or a derivative ora variant thereof (for example without the His tag or as shown in anyone of the sequences shown by SEQ ID NOs: 75-97) and the second bindingpartner (nominally Y) is a scFv or sdAb specific for GCN4 (for exampleas shown in SEQ ID NO: 3, 98 or 99) or a variant or a derivative or afragment thereof. In one embodiment, the first binding partner(nominally X) is a sFv or sdAb specific for GCN4 (for example as shownin SEQ ID NO: 3) or a variant or a derivative or a fragment thereof andthe second binding partner (nominally Y) is GCN4 (for example as shownin SEQ ID NO: 1) or a fragment or variant or a derivative thereof (forexample any of the sequences shown by SEQ ID NOs: 75-97).

GCN4 variants include an amino acid sequence with at least 80%, 85%,90%, 91%, 92%, 93%, 94% 95%, 96%, 97% or 98%, or 99% identity to SEQ IDNO: 1. GCN4 variants also include an amino acid having at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to a sequenceencoded by a nucleotide sequence SEQ ID NO: 2, or a sequence encoded bya nucleotide sequence which hybridises to SEQ ID NO: 2 under stringentconditions.

GCN4 fragments include amino acid sequences of GCN4 shorter than theamino acid sequence of SEQ ID NO: 1.

GCN4 derivatives refer to amino acid sequences of GCN4 longer, either atthe N terminal or at the C-terminal, than the amino acid sequence of SEQID NO: 1.

A suitable scFv specific to GCN4 is 52SR4 (SEQ ID NO: 3) or a variantthereof (SEQ ID NO: 98 or 99). Variants of 52SR4 include an amino acidsequence with at least 80%, or 85%, or 90%, or 95%, or 98%, or 99%identity to SEQ ID NO: 3. 52SR4 variants also include an amino acidsequence having at least at least 80%, or 85%, or 90%, or 95%, or 98%,or 99% to a sequence encoded by a nucleotide sequence SEQ ID NO: 4, or asequence encoded by a nucleotide sequence which hybridises to SEQ ID NO:4 under stringent conditions.

The present inventors have found that the single chain antibody 52SR4and peptide GCN4, are a binding pair suitable for use in the bispecificprotein complexes of the present disclosure.

Alternatively, any suitable antibody/fragment and antigen (such as apeptide) may be employed as X and Y. Preferably such an X and Y pairresult in greater than 75% heterodimer when A-X and Y-B are combined ina 1:1 molar ratio.

In one embodiment, the first binding partner (X) and the second bindingpartner(Y) are a protein.

In one embodiment, the first binding partner (X) is an enzyme or anactive fragment thereof and the second binding partner (Y) is a ligandor vice versa.

In one embodiment, the first binding partner (X) is an enzyme or anactive fragment thereof and the second binding partner (Y) is aninhibitor of that enzyme or vice versa.

“Active fragment” as employed herein refers to an amino acid fragment,which is less than the whole amino acid sequence for the entity andretains essentially the same biological activity or a relevantbiological activity, for example greater than 50% activity such as 60%,70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another embodiment, the first binding partner X is glutathione (GSH)and the second binding partner Y is glutathione-S-transferase (GST) orvice versa.

In another embodiment, X is Fos and Y is Jun or vice versa.

In another embodiment, X is His and Y is anti-His or vice versa.

In another embodiment, the binding pair is calmodulin binding peptideand Y is calmodulin or vice versa.

In another embodiment, X is maltose-binding protein and Y is ananti-maltose binding protein or fragment thereof or vice versa.

Other enzyme-ligand combinations are also contemplated for use inbinding partners. Also suitable are affinity tags known in the art forprotein purification because these have a tendency to bind with highaffinity to their respective binding partners.

“Identity”, as used herein, indicates that at any particular position inthe aligned sequences, the amino acid residue is identical between thesequences. “Similarity”, as used herein, indicates that, at anyparticular position in the aligned sequences, the amino acid residue isof a similar type between the sequences. For example, leucine may besubstituted for isoleucine or valine. Other amino acids which can oftenbe substituted for one another include but are not limited to:

-   -   phenylalanine, tyrosine and tryptophan (amino acids having        aromatic side chains);    -   lysine, arginine and histidine (amino acids having basic side        chains);    -   aspartate and glutamate (amino acids having acidic side chains);    -   asparagine and glutamine (amino acids having amide side chains);        and    -   cysteine and methionine (amino acids having sulphur-containing        side chains).

Degrees of identity and similarity can be readily calculated(Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing. Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, New York, 1991, the BLAST™software available from NCBI (Altschul, S. F. et al., 1990, J. Mol.Biol. 215:403-410; Gish, W. & States, D. J. 1993, Nature Genet.3:266-272. Madden, T. L. et al., 1996, Meth. Enzymol. 266:131-141;Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J.& Madden, T. L. 1997, Genome Res. 7:649-656,).

In one embodiment, the first or second binding partner (X or Y) is aprotein or peptide.

In one embodiment, the first and second fusion proteins comprise one ormore peptide linkers. The linkers may be incorporated at variouslocations in the fusion proteins. For example, a linker may beintroduced between a binding partner and the protein attached thereto.

In one embodiment, the linker is a peptide linker.

The term “peptide linker” as used herein refers to a peptide with anamino acid sequence. A range of suitable peptide linkers will be knownto the person of skill in the art.

In one embodiment, the binding partners of the bispecific proteincomplexes are joined to their respective proteins via peptide linkers.Examples of peptide linkers are shown in SEQ ID NOs; 5 to 74 (Tables 2,3 and 4).

In one embodiment the fusion proteins are a translational fusion, thatis a fusion protein expressed in a host cell comprising a geneticconstruct from which the fusion protein is expressed.

In one embodiment the fusion protein is prepared by fusing the heavychain of A to X and/or the heavy chain of B to Y optionally via apeptide linker.

In one embodiment, the peptide linker is 50 amino acids in length orless, for example 20 amino acids or less.

Generally it will be more efficient to express the fusion proteinrecombinantly and therefore a direct peptide bond or a peptide linkerthat can be expressed by a host cell may be advantageous.

In one embodiment, the linker is selected from a sequence shown insequence 5 to 72 or PPP.

TABLE 2 SEQ ID NO: SEQUENCE  5 DKTHTCAA  6 DKTHTCPPCPA  7DKTHTCPPCPATCPPCPA  8 DKTHTCPPCPATCPPCPATCPPCPA  9DKTHTCPPCPAGKPTLYNSLVMSDTAGTCY 10 DKTHTCPPCPAGKPTHVNVSVVMAEVDGTCY 11DKTHTCCVECPPCPA 12 DKTHTCPRCPEPKSCDTPPPCPRCPA 13 DKTHTCPSCPA

TABLE 3 SEQ ID NO: SEQUENCE 14 SGGGGSE 15 DKTHTS 16 (S)GGGGS 17(S)GGGGSGGGGS 18 (S)GGGGSGGGGSGGGGS 19 (S)GGGGSGGGGSGGGGSGGGGS 20(S)GGGGSGGGGSGGGGSGGGGSGGGGS 21 AAAGSG-GASAS 22 AAAGSG-XGGGS-GASAS 23AAAGSG-XGGGSXGGGS-GASAS 24 AAAGSG-XGGGSXGGGSXGGGS-GASAS 25AAAGSG-XGGGSXGGGSXGGGSXGGGS-GASAS 26 AAAGSG-XS-GASAS 27PGGNRGTTTTRRPATTTGSSPGPTQSHY 28 ATTTGSSPGPT 29 ATTTGS 30 AAAAAAAAAAAAA31 EPSGPISTINSPPSKESHKSP 32 GTVAAPSVFIFPPSD 33 GGGGIAPSMVGGGGS 34GGGGKVEGAGGGGGS 35 GGGGSMKSHDGGGGS 36 GGGGNLITIVGGGGS 37 GGGGVVPSLPGGGGS38 GGEKSIPGGGGS 39 RPLSYRPPFPFGFPSVRP 40 YPRSIYIRRRHPSPSLTT 41TPSHLSHILPSFGLPTFN 42 RPVSPFTFPRLSNSWLPA 43 SPAAHFPRSIPRPGPIRT 44APGPSAPSHRSLPSRAFG 45 PRNSIHFLHPLLVAPLGA 46 MPSLSGVLQVRYLSPPDL 47SPQYPSPLTLTLPPHPSL 48 NPSLNPPSYLHRAPSRIS 49 LPWRTSLLPSLPLRRRP 50PPLFAKGPVGLLSRSFPP 51 VPPAPVVSLRSAHARPPY 52 LRPTPPRVRSYTCCPTP- 53PNVAHVLPLLTVPWDNLR 54 CNPLLPLCARSPAVRTFP

(S) is optional in sequences 17 to 20. Another linker may be peptidesequence GS. Examples of rigid linkers include the peptide sequencesGAPAPAAPAPA (SEQ ID NO: 69), PPPP (SEQ ID NO: 70) and PPP.

Other linkers are shown in Table 4.

TABLE 4 SEQ ID NO: SEQUENCE 55 DLCLRDWGCLW 56 DICLPRWGCLW 57MEDICLPRWGCLWGD 58 QRLMEDICLPRWGCLWEDDE 59 QGLIGDICLPRWGCLWGRSV 60QGLIGDICLPRWGCLWGRSVK 61 EDICLPRWGCLWEDD 62 RLMEDICLPRWGCLWEDD 63MEDICLPRWGCLWEDD 64 MEDICLPRWGCLWED 65 RLMEDICLARWGCLWEDD 66EVRSFCTRWPAEKSCKPLRG 67 RAPESFVCYWETICFERSEQ 68 EMCYFPGICWM

In one aspect, there is provided a method of producing a bispecificprotein complex of the present disclosure, comprising the steps of:

-   -   (a) producing a first fusion protein (A-X), comprising a first        protein (A), attached to a first binding partner (X) of a        binding pair;    -   (b) producing a second fusion protein (B-Y), comprising a second        protein (B), attached to a second binding partner (Y) of a        binding pair; and    -   (c) mixing the first (A-X) and second fusion proteins (B-Y)        prepared in step a) and b) together.

Typically the mixing of A-X and B-Y in step (c) is in a 1:1 molar ratio.

In one embodiment each fusion protein employed in the complexes of thepresent disclosure is produced by expression in a host cell or hostcells in an expression experiment.

In one aspect, there is provided a method of preparing a bispecificprotein complex of the present disclosure, comprising the steps of:

-   (a) expressing a first fusion protein (A-X as defined herein),    comprising a first protein (A), attached to a first binding    partner (X) of a binding pair;-   (b) expressing a second fusion protein (B-Y as defined herein),    comprising a second protein (B), attached to a second binding    partner (Y) of a binding pair;

wherein fusion protein A-X and B—Y are expressed from the same host cellor distinct host cells.

Distinct host cells as employed herein refer to individual cells,including cells of the same type (even same clonal type).

In one embodiment the expression is transient expression. The use oftransient expression is highly advantageous when combined with theability to generate bispecific complexes without recourse topurification. This results in a rapid method to generate bispecificprotein complexes as transient transfection is much simpler and lessresource intensive than stable transfection.

In one embodiment the expression is stable expression i.e. wherein theDNA encoding the fusion protein in question is stably integrated intothe host cell genome.

In one embodiment a polynucleotide encoding A-X (as defined herein) anda polynucleotide encoding B-Y (as defined herein) on the same ordifferent polynucleotide sequences are transfected into a cell as partof a functional assay, wherein the proteins are expressed in the celland/or released therefrom. In particular the polynucleotides aretransiently transfected on the same of different plasmids.

The mixing of A-X and B—Y is generally effected in conditions where theX and Y can interact. In one embodiment, the fusion proteins areincubated in cell culture media under cell culturing conditions, forexample the fusion proteins are incubated for 90 minutes in a 37° C./5%CO₂ environment.

In one embodiment the fusion proteins of the present disclosure aremixed in an aqueous environment, for example one fusion protein may bebound to a solid surface such as a bead or a plate and the other fusionprotein can be introduced thereto in an aqueous solution/suspension. Thesolid phase allows excess components and reagents to be washed awayreadily. In one embodiment neither fusion is attached a solid phase andare simply mixed in a liquid/solution/medium. Thus in one embodiment A-Xand B—Y are mixed as free proteins in an aqueous media.

Advantageously, the method of the present disclosure can be employed toprepare complexes formed between heterogenous pairs (i.e. between thefirst fusion protein [A-X] and second fusion protein [B-Y]) whereininteractions between homogenous pairs (i.e. between two first fusionproteins [A-X] or two second fusion proteins [B-Y]) are minimised. Thusthe present method allows large numbers of bispecific protein complexesto be prepared, with minimal or no contamination with homodimericcomplexes. An advantage of the constructs and method of the presentdisclosure is that the ratio of A-X to B-Y is controlled by theproperties of the A-X and B—Y and in particular a molar ratio of 1:1 canbe achieved. This element of control is a significant improvement overthe certain prior art methods.

In one embodiment a method of the present disclosure comprises a furtherstep of transferring a pair of variable regions (in particular two pairsof variable regions) identified as having synergistic activity into analternative bispecific, trispecific or multispecific format, optionallyhumanising said variable regions if necessary beforehand, which is analternative therapeutic format and/or a format having an extendedhalf-life suitable for testing in assays with a longer duration (forexample which run a week or more).

“Multispecific” as used herein refers to antibodies or fragments thereofhaving at least two different binding sites each capable of binding toan epitope with different specificities, e.g. being able to cross-linktwo different antigens. Multispecific formats include those known in theart and those described herein, such as DVD-Igs, FabFvs for example asdisclosed in WO2009/040562 and WO2010/035012, diabodies, triabodies,tetrabodies etc.

Other examples of bi and multispecific formats (including therapeuticformats) include a diabody, triabody, tetrabody, tandem scFv, tandemscFv-Fc, FabFv, Fab′Fv, FabdsFv, Fab-scFv, Fab′-scFv, diFab, diFab′,scdiabody, scdiabody-Fc, ScFv-Fc-scFv, scdiabody-CH₃, IgG-scFv,scFv-IgG, V-IgG, IgG-V, DVD-Ig, and DuoBody.

Diabody as employed herein refers to two Fv pairs: VH/VL and a furtherVH/VL pair which have two inter-Fv linkers, such that the VH of a firstFv is linked to the VL of the second Fv and the VL of the first Fv islinked to the VH of the second Fv.

Triabody as employed herein refers to a format similar to the diabodycomprising three Fv pairs and three inter-Fv linkers.

Tetrabody as employed herein refers to a format similar to the diabodycomprising fours Fv pairs and four inter-Fv linkers.

Tandem scFv as employed herein refers to two scFvs (each comprising alinker is the usual manner) linked to each other via a single linkersuch that there is a single inter-Fv linker.

Tandem scFv-Fc as employed herein refers to two tandem scFvs, whereineach one is appended to the N-terminus of a CH2 domain, for example viaa hinge, of constant region fragment —CH2CH3.

FabFv as employed herein refers to a Fab fragment with a variable regionappended to the C-terminal of each of the following, the CH1 of theheavy chain and CL of the light chain. The format may be provided as aPEGylated version thereof.

Fab′Fv as employed herein is similar to FabFv, wherein the Fab portionis replaced by a Fab′. The format may be provided as a PEGylated versionthereof.

FabdsFv as employed herein refers to a FabFv wherein an intra-Fvdisulfide bond stabilises the appended C-terminal variable regions. Theformat may be provided as a PEGylated version thereof.

Fab-scFv as employed herein is a Fab molecule with a scFv appended onthe C-terminal of the light or heavy chain.

Fab′-scFv as employed herein is a Fab′ molecule with a scFv appended onthe C-terminal of the light or heavy chain.

DiFab as employed herein refers to two Fab molecules linked via theirC-terminus of the heavy chains.

DiFab′ as employed herein refers to two Fab′ molecules linked via one ormore disulfide bonds in the hinge region thereof.

As employed herein scdiabody is a diabody comprising an intra-Fv linker,such that the molecule comprises three linkers and forms a normal scFvwhose VH and VL terminals are each linked to a one of the variableregions of a further Fv pair.

Scdiabody-Fc as employed herein is two scdiabodies, wherein each one isappended to the N-terminus of a CH2 domain, for example via a hinge, ofconstant region fragment —CH2CH3. ScFv-Fc-scFv as employed herein refersto four scFvs, wherein one of each is appended to the N-terminus and theC-terminus of both the heavy and light chain of a —CH2CH3 fragment.Scdiabody-CH3 as employed herein refers to two scdiabody molecules eachlinked, for example via a hinge to a CH3 domain.

IgG-scFv as employed herein is a full length antibody with a scFv on theC-terminal of each of the heavy chains or each of the light chains.

scFv-IgG as employed herein is a full length antibody with a scFv on theN-terminal of each of the heavy chains or each of the light chains.

V-IgG as employed herein is a full length antibody with a variabledomain on the N-terminal of each of the heavy chains or each of thelight chains.

IgG-V as employed herein is a full length antibody with a variabledomain on the C-terminal of each of the heavy chains or each of thelight chains

DVD-Ig (also known as dual V domain IgG) is a full length antibody with4 additional variable domains, one on the N-terminus of each heavy andeach light chain.

Duobody or ‘Fab-arm exchange’ as employed herein is a bispecific IgGantibody format where matched and complementary engineered amino acidchanges in the constant domains (typically CH3) of two differentmonoclonal antibodies lead, upon mixing, to the formation ofheterodimers. A heavy/light chain pair from the first antibody will, asa result of the residue engineering, prefer to associate with aheavy:light chain pair of a second antibody.

If present constant region domains of a bispecific antibody complex orantibody molecule of the present disclosure, if present, may be selectedhaving regard to the proposed function of the complex or antibodymolecule, and in particular the effector functions which may berequired. For example, the constant region domains may be human IgA,IgD, IgE, IgG or IgM domains. In particular, human IgG constant regiondomains may be used, especially of the IgG1 and IgG3 isotypes when theantibody molecule is intended for therapeutic uses and antibody effectorfunctions are required. Alternatively, IgG2 and IgG4 isotypes may beused when the antibody molecule is intended for therapeutic purposes andantibody effector functions are not required. It will be appreciatedthat sequence variants of these constant region domains may also beused. For example IgG4 molecules in which the serine at position 241 hasbeen changed to proline as described in Angal et al., 1993, MolecularImmunology, 1993, 30:105-108 may be used. Accordingly, in the embodimentwhere the antibody is an IgG4 antibody, the antibody may include themutation S241P.

It will also be understood by one skilled in the art that antibodies mayundergo a variety of posttranslational modifications. The type andextent of these modifications often depends on the host cell line usedto express the antibody as well as the culture conditions. Suchmodifications may include variations in glycosylation, methionineoxidation, diketopiperazine formation, aspartate isomerization andasparagine deamidation. A frequent modification is the loss of acarboxy-terminal basic residue (such as lysine or arginine) due to theaction of carboxypeptidases (as described in Harris, R J. Journal ofChromatography 705:129-134, 1995). Accordingly, the C-terminal lysine ofthe antibody heavy chain may be absent.

The present disclosure also provides a composition comprising one ormore bispecific protein complexes as described above, wherein thecomposition predominantly comprises heterodimeric bispecific complexesaccording to the present disclosure, for example with minimal or nocontamination with homodimeric complexes.

In one embodiment, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 90%, or at least 95% of the fusion proteinsin the composition are in a bispecific protein complex form.

In one embodiment, at least 60% of the fusion proteins in thecomposition are in a bispecific protein complex form.

In one embodiment the complexes formed require no further purificationsteps and thus the compositions comprise unpurified bispecificcomplexes.

In one embodiment the complexes formed require one purification step,for example column chromatography.

In one embodiment the method further comprises at least one purificationstep, for example after expression of a fusion protein according to thepresent disclosure and before mixing the fusion proteins.

In one aspect the present disclosure relates to a fusion protein, aheterodimerically-tethered bispecific protein complex, a compositioncomprising a fusion protein or said bispecific protein complex, amultiple, array, library as defined herein.

In one embodiment, the bispecific protein complex is in solution orsuspension.

In one embodiment, the bispecific protein complexes are fixed on a solidsubstrate surface.

In one embodiment, the multiplex is in the form of an array, for examplein a microplate, such as a 96 or 384 well plate. Such arrays can bereadily implemented in screening assays to identify bispecific proteincomplexes with desired functionality.

In another embodiment, the bispecific protein complexes are conjugatedto beads.

A fusion protein as defined above is a component of the bispecificprotein complex according to the present disclosure. In one aspect, thepresent disclosure relates to a fusion protein described herein.

In a further aspect, there is provided a library, comprising two or morefusion proteins as defined above.

The term “library” as used herein refers to two or more bispecificantibody complexes of the present disclosure or multiple fusion proteinsof the present disclosure that can be combined to form at least twodifferent bispecific antibody complexes according to the presentdisclosure. As described throughout the specification, the term“library” is used in its broadest sense and may also encompasssub-libraries.

Advantageously, the library may comprise a range of different fusionproteins which have either the first binding partner (X) or secondbinding partner (Y) of a particular binding pair attached thereto. Inone embodiment part of the library comprisesproteins/antibodies/fragments each connected to a binding partner X andthe remainder of the library comprises the sameproteins/antibodies/fragments each connected to a binding partner Y.This thus allows any two fusion proteins to be readily combined to forma bispecific protein complex of the present disclosure, as long as onefusion protein has the first binding partner of a binding pair attachedand the other fusion protein has the second binding partner of thebinding pair attached.

In one embodiment bispecific protein complexes of the present inventionare suitable for therapeutic applications and may provide noveltherapies for treating diseases. Thus in a further aspect, there isprovided a bispecific protein complex as described above for use intherapy. The bispecific protein complex is suitable for treating a rangeof diseases, such as autoimmune disease and cancer.

Conversely, the bispecific protein complexes of the present disclosurecan be engineered with one antibody or antibody fragment specific forT-lymphocytes, and another antibody or antibody fragment specific for acancer-specific antigen. As a result, the bispecific antibody complexesof the present disclosure may advantageously possess a higher cytotoxicpotential compared to ordinary monoclonal antibodies.

The bispecific protein complexes of the present disclosure are alsoparticularly suited for inhibiting B cell function in order to controlimmune and autoimmune reactions in various autoimmune diseases.

Thus, the present disclosure extends to a method of treating a diseasein a patient, comprising the administration of a bispecific proteincomplex of the present disclosure.

In one aspect, there is provided a pharmaceutical composition comprisingone or more bispecific protein complexes of the present disclosure.

In one embodiment there is provided a fusion protein obtained orobtainable for a method of the present disclosure.

In one embodiment there is provided an bispecific antibody complexobtained or obtainable from a method of the present disclosure

In one embodiment there is provided a bispecific or multispecificantibody molecule comprising variable regions combinations identified bya method according to the present disclosure.

In one embodiment there is provided a composition, such as apharmaceutical composition comprising a fusion protein, a bispecificantibody complex or a bispecific/multispecific antibody moleculeobtained from a method of the present disclosure.

Various different components can be included in the composition,including pharmaceutically acceptable carriers, excipients and/ordiluents. The composition may, optionally, comprise further moleculescapable of altering the characteristics of the population of antibodiesof the invention thereby, for example, reducing, stabilizing, delaying,modulating and/or activating the function of the antibodies. Thecomposition may be in solid, or liquid form and may inter alia, be inthe form of a powder, a tablet, a solution or an aerosol.

The present disclosure also provides a pharmaceutical or diagnosticcomposition comprising a bispecific protein complex of the presentinvention in combination with one or more of a pharmaceuticallyacceptable excipient, diluent or carrier. Accordingly, provided is theuse of a bispecific protein complex of the invention for use in thetreatment and for the manufacture of a medicament for the treatment of apathological condition or disorder.

The pathological condition or disorder, may, for example be selectedfrom the group consisting of infections (viral, bacterial, fungal andparasitic), endotoxic shock associated with infection, arthritis such asrheumatoid arthritis, asthma such as severe asthma, chronic obstructivepulmonary disease (COPD), pelvic inflammatory disease, Alzheimer'sDisease, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, Peyronie's Disease, coeliac disease, gallbladder disease,Pilonidal disease, peritonitis, psoriasis, vasculitis, surgicaladhesions, stroke, Type I Diabetes, lyme disease, meningoencephalitis,autoimmune uveitis, immune mediated inflammatory disorders of thecentral and peripheral nervous system such as multiple sclerosis, lupus(such as systemic lupus erythematosus) and Guillain-Barr syndrome,Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave'sdisease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere'sdisease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma,Wegener's granulomatosis, other autoimmune disorders, pancreatitis,trauma (surgery), graft-versus-host disease, transplant rejection, heartdisease including ischaemic diseases such as myocardial infarction aswell as atherosclerosis, intravascular coagulation, bone resorption,osteoporosis, osteoarthritis, periodontitis, hypochlorhydia and cancer,including breast cancer, lung cancer, gastric cancer, ovarian cancer,hepatocellular cancer, colon cancer, pancreatic cancer, esophagealcancer, head & neck cancer, kidney, and cancer, in particular renal cellcarcinoma, prostate cancer, liver cancer, melanoma, sarcoma, myeloma,neuroblastoma, placental choriocarcinoma, cervical cancer, and thyroidcancer, and the metastatic forms thereof.

The present disclosure also provides a pharmaceutical or diagnosticcomposition comprising a bispecific protein complex of the presentinvention in combination with one or more of a pharmaceuticallyacceptable excipient, diluent or carrier. Accordingly, provided is theuse of a bispecific protein complex of the invention for use intreatment and in the manufacture of a medicament.

The composition will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. A pharmaceutical composition of the presentinvention may additionally comprise a pharmaceutically-acceptableadjuvant. The present invention also provides a process for preparationof a pharmaceutical or diagnostic composition comprising adding andmixing the antibody molecule or bispecific antibody complex of thepresent invention together with one or more of a pharmaceuticallyacceptable excipient, diluent or carrier.

The term “pharmaceutically acceptable excipient” as used herein refersto a pharmaceutically acceptable formulation carrier, solution oradditive to enhance the desired characteristics of the compositions ofthe present disclosure. Excipients are well known in the art and includebuffers (e.g., citrate buffer, phosphate buffer, acetate buffer andbicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride,liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensionscan be encapsulated in liposomes or biodegradable microspheres. Theformulation will generally be provided in a substantially sterile formemploying sterile manufacture processes.

This may include production and sterilization by filtration of thebuffered solvent solution used for the formulation, aseptic suspensionof the antibody in the sterile buffered solvent solution, and dispensingof the formulation into sterile receptacles by methods familiar to thoseof ordinary skill in the art.

The pharmaceutically acceptable carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolised macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragées, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

The bispecific protein complexes of the invention can be delivereddispersed in a solvent, e.g., in the form of a solution or a suspension.It can be suspended in an appropriate physiological solution, e.g.,physiological saline, a pharmacologically acceptable solvent or abuffered solution. Buffered solutions known in the art may contain 0.05mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to0.55 mg sodium citrate per 1 ml of water so as to achieve a pH of about4.0 to 5.0. As mentioned supra a suspension can made, for example, fromlyophilised antibody.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

The bispecific antibody complex (or bispecific/multispecific antibodymolecule of the present disclosure) may be the sole active ingredient inthe pharmaceutical or diagnostic composition or may be accompanied byother active ingredients including other antibody ingredients, forexample anti-TNF, anti-IL-1β, anti-T cell, anti-IFNγ or anti-LPSantibodies, or non-antibody ingredients such as xanthines. Othersuitable active ingredients include antibodies capable of inducingtolerance, for example, anti-CD3 or anti-CD4 antibodies.

In a further embodiment, the antibody, fragment or composition accordingto the disclosure is employed in combination with a furtherpharmaceutically active agent, for example a corticosteroid (such asfluticasone propionate) and/or a beta-2-agonist (such as salbutamol,salmeterol or formoterol) or inhibitors of cell growth and proliferation(such as rapamycin, cyclophosphmide, methotrexate) or alternatively aCD28 and/or CD40 inhibitor. In one embodiment the inhibitor is a smallmolecule. In another embodiment the inhibitor is an antibody specific tothe target.

The pharmaceutical compositions suitably comprise a therapeuticallyeffective amount of the bispecific antibody complex of the invention (ora bispecific/multispecific antibody molecule of the present disclosure).

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent needed to treat, ameliorate or prevent atargeted disease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any antibody, the therapeutically effectiveamount can be estimated initially either in cell culture assays or inanimal models, usually in rodents, rabbits, dogs, pigs or primates. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

The precise therapeutically effective amount for a human subject willdepend upon the severity of the disease state, the general health of thesubject, the age, weight and gender of the subject, diet, time andfrequency of administration, drug combination(s), reaction sensitivitiesand tolerance/response to therapy. This amount can be determined byroutine experimentation and is within the judgement of the clinician.Generally, a therapeutically effective amount will be from 0.01 mg/kg to50 mg/kg, for example 0.1 mg/kg to 20 mg/kg. Alternatively, the dose maybe 1 to 500 mg per day such as 10 to 100, 200, 300 or 400 mg per day.Pharmaceutical compositions may be conveniently presented in unit doseforms containing a predetermined amount of an active agent of theinvention.

Compositions may be administered individually to a patient or may beadministered in combination (e.g. simultaneously, sequentially orseparately) with other agents, drugs or hormones.

The dose at which the antibody molecule of the present invention isadministered depends on the nature of the condition to be treated, theextent of the inflammation present and on whether the antibody moleculeis being used prophylactically or to treat an existing condition. Thefrequency of dose will depend on the half-life of the antibody moleculeand the duration of its effect. If the antibody molecule has a shorthalf-life (e.g. 2 to 10 hours) it may be necessary to give one or moredoses per day. Alternatively, if the antibody molecule has a longhalf-life (e.g. 2 to 15 days) it may only be necessary to give a dosageonce per day, once per week or even once every 1 or 2 months.

In the present disclosure, the pH of the final formulation is notsimilar to the value of the isoelectric point of the antibody orfragment, for if the pH of the formulation is 7 then a pI of from 8-9 orabove may be appropriate. Whilst not wishing to be bound by theory it isthought that this may ultimately provide a final formulation withimproved stability, for example the antibody or fragment remains insolution.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, seeWO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a specific tissue ofinterest. Dosage treatment may be a single dose schedule or a multipledose schedule. Where the product is for injection or infusion, it maytake the form of a suspension, solution or emulsion in an oily oraqueous vehicle and it may contain formulatory agents, such assuspending, preservative, stabilising and/or dispersing agents.Alternatively, the bispecific protein complex (orbispecific/multispecific antibody molecule of the present disclosure)may be in dry form, for reconstitution before use with an appropriatesterile liquid. If the composition is to be administered by a routeusing the gastrointestinal tract, the composition will need to containagents which protect the antibody from degradation but which release thebispecific protein complex once it has been absorbed from thegastrointestinal tract.

A nebulisable formulation according to the present disclosure may beprovided, for example, as single dose units (e.g., sealed plasticcontainers or vials) packed in foil envelopes. Each vial contains a unitdose in a volume, e.g., 2 ml, of solvent/solution buffer.

The term “variant” as used herein refers to peptide or protein thatcontains at least one amino acid sequence or nucleotide sequencealteration as compared to the amino acid or nucleotide sequence of thecorresponding wild-type peptide or protein. A variant may comprise atleast 80%, or 85%, or 90%, or 95%, or 98% or 99% sequence identity tothe corresponding wild-type peptide or protein. However, it is possiblefor a variant to comprise less than 80% sequence identity, provided thatthe variant exhibits substantially similar function to its correspondingwild-type peptide or protein.

Antigens include cell surface receptors such as T cell or B cellsignalling receptors, co-stimulatory molecules, checkpoint inhibitors,natural killer cell receptors, Immunolglobulin receptors, TNFR familyreceptors, B7 family receptors, adhesion molecules, integrins,cytokine/chemokine receptors, GPCRs, growth factor receptors, kinasereceptors, tissue-specific antigens, cancer antigens, pathogenrecognition receptors, complement receptors, hormone receptors orsoluble molecules such as cytokines, chemokines, leukotrienes, growthfactors, hormones or enzymes or ion channels, epitopes, fragments andpost translationally modified forms thereof.

In one embodiment, the bispecific protein complex comprises one or twocell surface receptor specificities.

In one embodiment, the bispecific protein complex comprises one or twocytokine or chemokine specificities.

Antibodies or fragments to a pair of targets identified by the methodaccording to the present disclosure may be incorporated into any formatsuitable for use as a laboratory reagent, an assay reagent or atherapeutic.

Thus in one aspect the disclosure extends to use of antibodies fragmentsor combinations thereof as pairs in any format, examples of which aregiven above.

The disclosure also extends to compositions, such as pharmaceuticalcompositions comprising said novel formats with the particular antigenspecificity.

In a further aspect the disclosure includes use of the formats and thecompositions in treatment.

In one embodiment, the bispecific protein complex of the presentdisclosure may be used to functionally alter the activity of the antigenor antigens of interest. For example, the bispecific protein complex mayneutralize, antagonize or agonise the activity of said antigen orantigens, directly or indirectly.

The present disclosure also extends to a kit, for example comprising:

-   a) one or more fusion proteins (A-X as defined herein) comprising a    first antibody or antibody fragment (A) attached to a first binding    partner of a binding pair (X); and-   b) one or more fusion proteins (B-Y as defined herein) comprising a    second antibody or antibody fragment (B) attached to a second    binding partner of the binding pair (Y), wherein the latter is    specific for the first binding partner;    -   for example wherein the first binding partner (X) is a peptide        or polypeptide and the second binding (Y) partner is an antibody        or antibody fragment specific thereto;

wherein Y the second binding partner is specific to the first bindingpartner X and the second binding partner is, for example an antibody orantibody fragment specific thereto; and the specific interaction (suchas a binding interaction) of the two binding partners forms aheterodimer-tether which physically brings the two fusion proteins froma) and b) together to form a bispecific protein complex; and

wherein the fusion protein(s) is/are in a complexed or a non-complexedform.

Advantageously, the kit may comprise bispecific protein complexes of thepresent disclosure, or may comprise fusion proteins which are in acomplexed or non-complexed form. In the former case, the bispecificprotein complexes are ready for use “out of the box” which providesconvenience and ease of use, whereas in the latter case, the bispecificprotein complexes can be assembled according to the user's requirementsby combining different fusion proteins.

In another embodiment, the kit further comprises instructions for use.

In yet another embodiment, the kit further comprises one or morereagents for performing one or more functional assays.

In one embodiment, fusion proteins, bispecific proteins complexes,multiplexes, grids, libraries, compositions etc as described herein arefor use as a laboratory reagent.

In a further aspect, there is provided a nucleotide sequence, forexample a DNA sequence encoding a fusion protein and/or a bispecificprotein complex as defined above.

In one embodiment, there is provided a nucleotide sequence, for examplea DNA sequence encoding a bispecific protein complex according to thepresent disclosure.

In one embodiment there is provided a nucleotide sequence, for example aDNA sequence encoding a bispecific or multispecific antibody moleculeaccording to the present disclosure. The disclosure herein also extendsto a vector comprising a nucleotide sequence as defined above.

The term “vector” as used herein refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. An example of a vector is a “plasmid,” which is a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell, where they are subsequently replicated along with the hostgenome. In the present specification, the terms “plasmid” and “vector”may be used interchangeably as a plasmid is the most commonly used formof vector.

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbor Publishing.

The term “selectable marker” as used herein refers to a protein whoseexpression allows one to identify cells that have been transformed ortransfected with a vector containing the marker gene. A wide range ofselection markers are known in the art. For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. The selectable marker can also be a visuallyidentifiable marker such as a fluorescent marker for example. Examplesof fluorescent markers include rhodamine, FITC, TRITC, Alexa Fluors andvarious conjugates thereof.

Also provided is a host cell comprising one or more cloning orexpression vectors comprising one or more DNA sequences encoding anantibody of the present disclosure. Any suitable host cell/vector systemmay be used for expression of the DNA sequences encoding the antibodymolecule of the present disclosure. Bacterial, for example E. coli, andother microbial systems may be used or eukaryotic, for examplemammalian, host cell expression systems may also be used. Suitablemammalian host cells include CHO, myeloma or hybridoma cells.

The present disclosure also provides a process for the production of afusion protein according to the present disclosure comprising culturinga host cell containing a vector of the present disclosure underconditions suitable for leading to expression of protein from DNAencoding the molecule of the present disclosure, and isolating themolecule.

The bispecific protein complexes of the present disclosure may be usedin diagnosis/detection kits, wherein bispecific protein complexes withparticular combinations of antigen specificities are used. For example,the kits may comprise bispecific antibody complexes that are specificfor two antigens, both of which are present on the same cell type, andwherein a positive diagnosis can only be made if both antigens aresuccessfully detected. By using the bispecific antibody complexes of thepresent disclosure rather than two separate antibodies or antibodyfragments in a non-complexed form, the specificity of the detection canbe greatly enhanced.

In one embodiment, the bispecific antibody complexes are fixed on asolid surface. The solid surface may for example be a chip, or an ELISAplate.

Further provided is the use of a bispecific protein complex of thepresent disclosure for detecting in a sample the presence of a first anda second peptide, whereby the bispecific complexes are used as detectionagents.

The bispecific antibody complexes of the present disclosure may forexample be conjugated to a fluorescent marker which facilitates thedetection of bound antibody-antigen complexes. Such bispecific antibodycomplexes can be used for immunofluorescence microscopy. Alternatively,the bispecific antibody complexes may also be used for western blottingor ELISA.

In one embodiment, there is provided a process for purifying an antibody(in particular an antibody or fragment according to the invention).

In one embodiment, there is provided a process for purifying a fusionprotein or bispecific protein complex according the present disclosurecomprising the steps: performing anion exchange chromatography innon-binding mode such that the impurities are retained on the column andthe antibody is maintained in the unbound fraction. The step may, forexample be performed at a pH about 6-8.

The process may further comprise an initial capture step employingcation exchange chromatography, performed for example at a pH of about 4to 5.

The process may further comprise of additional chromatography step(s) toensure product and process related impurities are appropriately resolvedfrom the product stream.

The purification process may also comprise of one or moreultra-filtration steps, such as a concentration and diafiltration step.

“Purified form” as used supra is intended to refer to at least 90%purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure.

In the context of this specification “comprising” is to be interpretedas “including”.

Aspects of the disclosure comprising certain elements are also intendedto extend to alternative embodiments “consisting” or “consistingessentially” of the relevant elements. Positive embodiments employedherein may serve basis for the excluding certain aspects of thedisclosure.

Disclosures in the context of the method relating to the bispecificcomplexes apply equally to the complexes per se and vice versa.

All references referred to herein are specifically incorporated byreference.

REFERENCES

-   1. Ribosome display efficiently selects and evolves high-affinity    antibodies in vitro from immune libraries. Hanes J, Jermutus L,    Weber-Bornhauser S, Bosshard H R, Plückthun A. (1998) Proc. Natl.    Acad. Sci. U.S.A. 95, 14130-14135-   2. Directed in Vitro Evolution and Crystallographic Analysis of a    Peptide-binding Single Chain Antibody Fragment (scFv) with Low    Picomolar Affinity. Zhand C, Spinelli S, Luginbuhl B, Amstutz P,    Cambillau C, Pluckthun A. (2004) J. Biol. Chem. 279, 18870-18877-   3. Antigen recognition by conformational selection. Berger C,    Weber-Bornhauser S, Eggenberger Y, Hanes J, Pluckthun A,    Bosshard H. R. (1999) F.E.B.S. Letters 450, 149-153

EXAMPLES Example 1: Evaluation of Heterodimerically Tethered ProteinComplexes to Bispecific Antibody Targets with V Regions Derived fromDifferent Methods Combined in Different formats

Introduction:

For screening large numbers of bispecific combinations, V regions may bederived from different methods & hence be linked by a heterodimerictether in different A-X:Y-B formats. For example V regions derived by Bcell culture & single cell isolations may be in a Fab-X or Fab-Y formatto generate Fab-X:Fab-Y bispecific. V regions derived by phage displaymay be in a scFv-X or scFv-Y format to generate scFv-X:scFv-Ybispecific. Bispecifics may also be generated by combining differentlysourced V regions & formats ie Fab-X:scFv:Y or scFv:X:Fab-Y. Fab-X,Fab-Y, scFv-X or scFv-Y may be purified or utilised as a quantitatedtransient supernatant. In this example V regions derived by B cellculture & single cell isolations were combined with each other(Fab-X:Fab-Y) and with V regions derived from Phage display(Fab-X:scFv-Y). X in the examples herein below denotes the scFv 52SR4(SEQ ID NO:3) and Y is the GCN4 peptide (SEQ ID NO:1).

Methods:

Immunisation:

DNA encoding selected antigens was obtained by gene synthesis orcommercial sources & cloned into an expression vector with a strongconstitutive promoter. Plasmid DNA was then transfected into Rab-9rabbit fibroblast cells (ATCC® CRL-1414™) using an in-houseelectroporation system. Twenty four hours later cells were checked forantigen expression by flow cytometry & frozen in aliquots in liquidnitrogen until use. Up to 6 antigens were immunised per rabbit by eitherco-expression on the same cell or making mixtures of singly or multipletransfected cells. Rabbits were immunised with 3 doses of cells.

Antibody Discovery by B Cell Culture & Isolation:

B cell cultures were prepared using a method similar to that describedby Zubler et al. (1985). Briefly, spleen or PBMC-derived B cells fromimmunized rabbits were cultured at a density of approximately 2000-5000cells per well in bar-coded 96-well tissue culture plates with 200μl/well RPMI 1640 medium (Gibco BRL) supplemented with 10% FCS (PAAlaboratories ltd), 2% HEPES (Sigma Aldrich), 1% L-Glutamine (Gibco BRL),1% penicillin/streptomycin solution (Gibco BRL), 0.1% 0-mercaptoethanol(Gibco BRL), 3% activated splenocyte culture supernatant andgamma-irradiated mutant EL4 murine thymoma cells (5×10⁴/well) for sevendays at 37° C. in an atmosphere of 5% CO₂.

The presence of antigen-specific antibodies in B cell culturesupernatants was determined using a homogeneous fluorescence-basedbinding assay using HEK293 cells co-transfected with the antigens thatthe rabbits were immunized with. Screening involved the transfer of 10ul of supernatant from barcoded 96-well tissue culture plates intobarcoded 384-well black-walled assay plates containing HEK293 cellstransfected with target antigen (approximately 3000 cells/well) using aMatrix Platemate liquid handler. Binding was revealed with a goatanti-rabbit IgG Fcγ-specific Cy-5 conjugate (Jackson). Plates were readon an Applied Biosystems 8200 cellular detection system.

Following primary screening, positive supernatants were consolidated on96-well bar-coded master plates using an Aviso Onyx hit-picking robotand B cells in cell culture plates frozen at −80° C. Master plates werethen screened in a homogeneous fluorescence-based binding assay onHEK293 cells transfected with antigens separately and Superavidin™ beads(Bangs Laboratories) coated with recombinant protein as a source ofantigen. This was done in order to determine the antigen specificity foreach well.

To allow recovery of antibody variable region genes from a selection ofwells of interest, a deconvolution step was performed to enableidentification of the antigen-specific B cells in a given well thatcontained a heterogeneous population of B cells. This was achieved usingthe Fluorescent foci method (Clargo et al., 2014.Mabs 2014 Jan. 1: 6(1)143-159; EP1570267B1). Briefly, Immunoglobulin-secreting B cells from apositive well were mixed with either HEK293 cells transfected withtarget antigen or streptavidin beads (New England Biolabs) coated withbiotinylated target antigen and a 1:1200 final dilution of a goatanti-rabbit Fcγ fragment-specific FITC conjugate (Jackson). After staticincubation at 37° C. for 1 hour, antigen-specific B cells could beidentified due to the presence of a fluorescent halo surrounding that Bcell. A number of these individual B cell clones, identified using anOlympus microscope, were then picked with an Eppendorf micromanipulatorand deposited into a PCR tube. The fluorescent foci method was also usedto identify antigen-specific B cells from a heterogeneous population ofB cells directly from the bone marrow of immunized rabbits.

Antibody variable region genes were recovered from single cells byreverse transcription (RT)-PCR using heavy and light chain variableregion-specific primers. Two rounds of PCR were performed, with thenested secondary PCR incorporating restriction sites at the 3′ and 5′ends allowing cloning of the variable region into mouse Fab-X and Fab-Y(VH) or mouse kappa (VL) mammalian expression vectors. Heavy and lightchain constructs for the Fab-X and Fab-Y expression vectors wereco-transfected into HEK-293 cells using Fectin 293 (Life Technologies)or Expi293 cells using Expifectamine (Life Technologies) and recombinantantibody expressed in 6-well tissue culture plates in a volume of 5 ml.After 5-7 days expression, supernatants were harvested. Supernatantswere tested in a homogeneous fluorescence-based binding assay on HEK293cells transfected with antigen and Superavidin™ beads (BangsLaboratories) coated with recombinant protein or antigen transfected HEKcells. This was done to confirm the specificity of the clonedantibodies.

Production of Small Scale Fab A-X and Fab B-Y

The Expi293 cells were routinely sub-cultured in Expi293™ ExpressionMedium to a final concentration of 0.5×10⁶ viable cells/mL and wereincubated in an orbital shaking incubator (Multitron, Infors HT) at 120rpm 8% CO₂ and 37° C.

On the day of transfection cell viability and concentration weremeasured using an automated Cell Counter (Vi-CELL, Beckman Coulter). Toachieve a final cell concentration of 2.5×10⁶ viable cells/mL theappropriate volume of cell suspension was added to a sterile 250 mLErlenmeyer shake flask and brought up to the volume of 42.5 mL by addingfresh, pre-warmed Expi293™ Expression Medium for each 50 mLtransfection.

To prepare the lipid-DNA complexes for each transfection a total of 50μs of heavy chain and light chain plasmid DNAs were diluted in Opti-MEM®I medium (LifeTechnologies) to a total volume of 2.5 mL and 135 μL ofExpiFectamine™ 293 Reagent (LifeTechnologies) was diluted in Opti-MEM® Imedium to a total volume of 2.5 mL. All dilutions were mixed gently andincubate for no longer than 5 minutes at room temperature before eachDNA solution was added to the respective diluted ExpiFectamine™ 293Reagent to obtain a total volume of 5 mL. The DNA-ExpiFectamine™ 293Reagent mixtures were mixed gently and incubated for 20-30 minutes atroom temperature to allow the DNA-ExpiFectamine™ 293 Reagent complexesto form.

After the DNA-ExpiFectamine™ 293 reagent complex incubation wascompleted, the 5 mL of DNA-ExpiFectamine™ 293 Reagent complex was addedto each shake flask. The shake flasks were incubated in an orbitalshaking incubator (Multitron, Infors HT) at 120 rpm, 8% CO₂ and 37° C.

Approximately 16-18 hours post-transfection, 250 μL of ExpiFectamine™293 Transfection Enhancer 1 (LifeTechnologies) and 2.5 mL ofExpiFectamine™ 293 Transfection Enhancer 2 (LifeTechnologies) were addedto each shake flask.

The cell cultures were harvested 7 days post transfection. The cellswere transferred into 50 mL spin tubes (Falcon) and spun down for 30 minat 4000 rpm followed by sterile filtration through a 0.22 um Stericup(Merck Millipore). The clarified and sterile filtered supernatants werestored at 4° C. Final expression levels were determined by ProteinG-HPLC.

Small Scale (50 ml) Purification:

Both Fab-X and Fab-Y were purified separately by affinity capture usinga small scale vacuum based purification system. Briefly, the 50 ml ofculture supernatants were 0.22 μm sterile filtered before 500 μL of NiSepharose beads (GE Healthcare) were added. The supernatant beadsmixture was then tumbled for about an hour before supernatant wasremoved by applying vacuum. Beads were then washed with Wash 1 (50 mMSodium Phosphate 1 M NaCl pH 6.2) and Wash 2 (0.5 M NaCl). Elution wasperformed with 50 mM sodium acetate, pH4.0+1M NaCl. The eluted fractionsbuffer exchanged into PBS (Sigma), pH7.4 and 0.22 μm filtered. Finalpools were assayed by A280 scan, SE-UPLC (BEH200 method), SDS-PAGE(reduced & non-reduced) and for endotoxin using the PTS Endosafe system.

Antibody Discovery by Phage Display:

Phage selections were completed using an in-house large naive human scFvphagemid display library. Antigens were either expressed on Rab-9 rabbitfibroblast cells (ATCC® CRL1414™) using an in-house electroporationsystem, or transiently expressed on HEK293 cells using Fectin 293 (LifeTechnologies), or displayed as recombinant Fc fusions from R&D systems(1968-SL, 1430-CD) directly coated onto Nunc maxisorb ELISA plates.

For cell panning ˜10¹² phages, ˜1-10×10⁷ antigen transfected cells and˜1-10×10⁷ non-antigen transfected cells were blocked in PBS containing3% BSA, 0.5 mM EDTA, 0.1% Sodium Azide. Blocked phage particles werethen incubated with blocked non-antigen transfected cells for at least30 minutes, shaking on ice. Unbound phage were then removed fromnon-antigen transfected cells and incubated with blocked antigentransfected cells for at least 30 minutes, shaking on ice. Thetransfected cells were then washed four times in cold PBS containing 0.5mM EDTA to remove any unbound phage. Phage which had bound to antigenthe transfected cells was then eluted using 100 mM HCl and neutralisedwith 1 M Tris-HCl (pH 7.4). The cell debris was re-suspended in PBS, andboth cell debris and acid neutralised eluate were used to infectexponentially-growing (˜0.5 OD₆₀₀) E. coli TG-1 cells (Lucigen), at 37°C. for 1 hour. TG-1 cells were then plated on agar containing antibioticselective media and grown overnight at 30° C.

For Nunc maxisorb ELISA plate panning, plates were coated withrecombinant Fc fusions from R&D systems (1968-SL, 1430-CD) at 5 μg/mLovernight at 5° C. in PBS. The next day ˜10¹² phages were blocked in PBScontaining 1.5% BSA and 2.5% skimmed milk powder for at least one hour.ELISA plates were blocked in 3% BSA for at least one hour. Blocked phageparticles were added to washed blocked ELISA plates for at least 30minutes. Unbound phages were then removed by washing in PBS containing0.1% Tween 20. Phage which had bound to the ELISA plates was then elutedusing 100 mM HCl and neutralised with 1 M Tris-HCl (pH 7.4). The acidneutralised eluate was used to infect exponentially-growing (˜0.5 OD₆₀₀)E. coli TG-1 cells, at 37° C. for 1 hour. TG-1 cells were then plated onagar containing antibiotic selective media and grown overnight at 30° C.

Three rounds of panning were completed for each experiment of justcells, just protein or a combination of the two techniques. Between eachround phage particles were rescued by the following method.

Phagemid-containing TG1 cells (approximately 5×10⁹ in total) were usedto inoculate 2×TY (containing antibiotics and 0.1% glucose) and grown at37° C., 250 rpm, until they reach mid-log phase, OD₆₀₀=0.5-0.6, at whichpoint they were infected with M13KO7 (Amersham-Pharmacia) interferenceresistant helper phage (20-fold MOI). The culture was swirled and leftto stand for thirty minutes at 37° C., followed by a slow shake, 50 rpm,at 37° C. for thirty minutes. The helper phage-infected TG1 cells werethen pelleted by centrifugation at 2,500×g for 15 minutes, thesupernatant was removed and the cells were resuspended in 2×TY(containing antibiotics). Resuspended cells were grown for 16 hours at30° C. shaking at 250 rpm, to allow phage production.

The following morning, the cells were separated from the culture bycentrifugation at 2,500×g for 15 minutes. The phage-containingsupernatant was removed into a fresh tube and the centrifugationrepeated. To the purified supernatant, a fifth volume of 20% PEG salt(2.5M NaCl, 20% (w/v) PEG 8000) was added, mixed and left on ice forthirty minutes, to precipitate phage from the supernatant. Theprecipitated phages were then pelleted by centrifugation at 2,500×g for15 minutes, and the phage pellet was resuspended in PBS ready for thenext round of panning.

Following the completion of three rounds of panning, plasmidpurification and concatenate 1 mL scale phage rescues were performed forindividual E. coli colonies picked from output phage infected coloniesfrom the final round of panning. Where possible each sample was testedfor binding recombinant Fc fusions or irrelevant protein by ELISA. Apositive ‘hit’ was determined to be any sample with an antigen bindingsignal greater than three times the negative signal. Briefly all ELISAplates were coated overnight at 4° C. with antigen at 2 μg/ml in PBS.Washes were performed between each step of the assay, and consisted offour washes in PBS (containing 0.1% Tween20). All plates were blocked inPBS containing 3% BSA, and all samples were blocked in PBS containing2.5% milk, for at least an hour prior to addition of screening samplesto ELISA plates. Anti-M13 HRP (GE Healthcare) was then added at a 1 in5,000 dilution in PBS (containing 3% BSA), for 1 hour. Following thefinal wash, TMB (Calbiochem) was added to all wells, and the OD ofplates was read at 630 and 490 nm, with AOD recorded using a BiotekSynergy plate reader. A selection of hits from different panningstrategies where sub-cloned from the phagemid vector to a mammalianexpression vector to generate scFv-FC constructs. Where recombinantprotein was not available for screening scFv's were sub-cloned withoutprior screening. ScFv-FC constructs were transfected into HEK-293 cellsusing Fectin 293 (Life Technologies) or Expi293 cells usingExpifectamine (Life Technologies) and recombinant antibody expressed in6-well tissue culture plates in a volume of 5 ml. After 5-7 daysexpression, supernatants were harvested. The presence ofantigen-specific antibodies in HEK293 culture supernatants wasdetermined using a homogeneous fluorescence-based binding assay usingHEK293 cells co-transfected with the antigens that the scFvs were pannedagainst. Screening involved the transfer of 10 μl of supernatant intobarcoded 384-well black-walled assay plates containing HEK293 cellstransfected with target antigen (approximately 3000 cells/well). Bindingwas revealed with a goat anti-mouse IgG Fcγ-specific Cy-5 conjugate(Jackson). Plates were read on an Applied Biosystems 8200 cellulardetection system. This was done to confirm the specificity of the clonedantibodies.

In order for the scFv's to be tested for functional activity in thebioassay they were sub-cloned again into the scFv-Y mammalian expressionvector (AAASGGG linker) as well as Fab-X (ASGGGG linker) and Fab-Y(ASGGG linker) (VH) or mouse kappa (VL) mammalian expression vectors.

Mammalian expression vector for scFv-Y format shown in FIG. 7.

Reagent Supplier Catalogue number Anti-M13 HRP GE Healthcare 27942101Human CD22-Fc chimera R&D Systems 1968-SL Human CD45-Fc chimera R&DSystems 1430-CD 100 mM HCl Sigma 2104-50ML 1M Trizma-hydrochloride soln.Sigma T2694 - 100ML (pH 8) Anti-Mouse IgG Feγ-specific Cy-5 Jackson115-606-008 Expifectamine transfection Kit Life Technologies A14524 KODhot start polymerase MerckMillipore 71086 Freestyle 293 expressionmedium Life Technologies 12338-018 293fectin transfection reagent LifeTechnologies 12347-500 Phosphate Buffer Saline (PBS) Fisher Scientific10562765 EDTA Sigma 03690 Sodium Azide (NaN3) Sigma S2002 Bovine SerumAlbumin (BSA) Sigma A1470 Skimmed milk powder Sigma 70166 PEG 8,000 AlfaAeser 43443 Sodium Chloride VWR 55011433

Screening Assays

Donor PBMCs were rapidly thawed using a water bath set to 37° C., andcarefully transferred to a 50 ml Falcon tube. They were then diluteddropwise to 5 ml in assay media to minimise the osmotic shock. The cellswere then diluted to 20 ml carefully before adding the final mediadiluent to make the volume 50 ml. The cells were then spun at 500 g for5 minutes before removing the supernatant and resuspending the cells in1 ml media. The cells were then counted and diluted to 1.66×10⁶ cells/mlbefore dispensing 30 μl per well into a V-bottom TC plate giving a finalassay concentration of 5.0×10⁴ cells/well. The cell plate was thenstored covered in a 37° C., 5% CO₂ incubator until they were required,giving them a minimum of 1 hour to rest.

Fab-X and Fab-Y reagents were mixed in an equimolar ratio at 5× thefinal assay concentration in assay media and incubated for 90 min at 37°C., 5% CO₂. Samples were prepared in a 96-well U-bottom polypropyleneplate and covered during the incubation. 10 μl of 5× Fab or scFv-X+Fabor scFv-Y mixture was added to the appropriate test wells containingcells and mixed by shaking at 1000 rpm for 30 sec prior to beingincubated for 90 min at 37° C., 5% CO₂.

The cells were then stimulated with 10 μl of anti-human IgM. The finalassay concentration of stimulus varied depending on the assay panelreadouts, the three antibody cocktails A, B and C (detailed below) werestimulated at a final assay concentration of either 50 μg/ml (cocktail A& C) or 25 μg/ml (cocktail B). The assay plates were then gently mixedat 1000 rpm for 30 sec prior to incubation at 37° C., 5% CO₂ for 5 min(antibody cocktail A & C) or 2 min (antibody cocktail B). The assay wasstopped by adding 150 μl ice-cold BD CytoFix to all wells and incubatedfor 15 min at RT. The fixed cells were then spun at 500 g for 5 min topellet the cells and allow removal of the supernatant using a BioTekELx405 plate washer. The pellet was re-suspended by vortexing the plateat 2400 rpm for 30 sec. The cells were then permeabilised at 4° C. byadding 100 μl ice-cold BD Cell Permeabilisation Buffer III for 30 min.The cells were then washed in 100 μl FACS buffer and spun at 500 g for 5min. Supernatant was again removed by the ELx405 before using it torapidly dispense 200 μl FACS Buffer to wash away any residualpermeabilisation buffer. Cells were again spun at 500 g and thesupernatant removed by inversion. During the preceding spin step theantibody cocktail was prepared in FACS Buffer and kept shielded from thelight. The cells were then re-suspended by vortexing (2400 RPM, 30 sec)before 20 μl of antibody cocktail was added to all wells and the plateshaken for 30 sec at 1000 rpm. The cells were then incubated for 60 minat RT in the dark.

The cells were then washed twice in 200 μl FACS buffer with a 500 g spinand supernatant removed after each step. Finally the cells werere-suspended by vortexing for 30 sec at 2400 rpm before adding a final20 μl FACS buffer. The plate(s) were then read on the IntellicytHTFC/iQue instrument.

FACS Buffer=PBS+1% BSA+0.05% NaN₃+2 mM EDTA

Antibody Cocktail=1:5 CD20 PE (BD Biosciences)+1:5 PLCγ2 AF88+1:10 AktAF647 (diluted in FACS buffer).

Reagent Supplier Catalogue number Anti-human IgM Southern Biotech2022-14 CytoFix BD Biosciences 554655 Perm Buffer III BD Biosciences558050 Anti Akt (pS473) AF647 BD Biosciences 561670 Anti PFCγ2 (pY759)AF488 BD Biosciences 561174 Anti-human CD20 PE BD Biosciences 558021Phosphate Buffer Saline (PBS) Fisher Scientific 10562765 RPMI 1640 LifeTechnologies 31870 Foetal Calf Serum (FCS) Life Technologies 16140Glutamax Life Technologies 35050 Penicillin/Streptomycin (P/S) LifeTechnologies 15070 EDTA Sigma 03690 Sodium Azide (NaN3) Sigma S2002Bovine Serum Albumin (BSA) Sigma A1470

Data was captured and evaluated using commercially available softwaretools.

Results

FIG. 8 and FIG. 9 demonstrate the inhibition of phosphorylation of PLCγ2and Akt in human B cells when activated with anti-IgM on treatment withCD79b/CD22 and CD79b/CD45 bispecific combination formed byheterodimerically tethered Fab-X+Fab-Y or Fab-X+scFv-Y constructs. TheFab-X and Fab-Y constructs were purified and the scFv-Y constructs wereunpurified transient HEK supernatants.

1. A bispecific protein complex having the formula A-X:Y-B wherein: A-Xis a first fusion protein; Y-B is a second fusion protein; X:Y is aheterodimeric-tether; : is a binding interaction between X and Y; A is afirst protein component of the bispecific protein complex independentlyselected from the group comprising a Fab fragment, a Fab′ fragment,single domain antibody (sdAb) and a single chain Fv (scFv); B is asingle chain Fv or sdAb; X is a first binding partner of a binding pairindependently selected from an antigen, a Fab fragment, a Fab′ fragment,a single chain Fv and sdAb; and Y is a second binding partner of thebinding pair independently selected from antigen, a Fab fragment, a Fab′fragment, a single chain Fv and a sdAb; with the proviso that when X isan antigen Y is a Fab fragment, a Fab′ fragment, a single chain Fv or asdAb specific to the antigen represented by X and when Y is an antigen Xis a Fab fragment, a Fab′ fragment, a single chain Fv or a sdAb specificto the antigen represented by Y.
 2. The bispecific protein complexaccording to claim 1, wherein X is fused to the C-terminal of A, or tothe C-terminus of the scFv or sdAb.
 3. The bispecific protein complexaccording to claim 1, wherein Y is fused to the C-terminal of B.
 4. Thebispecific protein complex according to claim 1, wherein the bindingaffinity between X and Y is 5 nM or stronger.
 5. (canceled)
 6. Thebispecific protein complex according to claim 1, wherein A is a Fabfragment.
 7. The bispecific protein complex according to claim 6,wherein X is independently selected from a Fab fragment, a Fab′fragment, a scFv, a sdAb and an antigen with the proviso that when X isa Fab fragment, a Fab′ fragment, a scFv or sdAb then Y is an antigen andwhen X is an antigen Y is a Fab fragment, a Fab′ fragment, a scFv or asdAb.
 8. The bispecific protein complex according to claim 7, wherein Xis a Fab fragment, a Fab′ fragment, a scFv or sdAb.
 9. The bispecificprotein complex according to claim 8 wherein the Fab fragment, the Fab′fragment, the scFv or the sdAb is specific to the peptide GCN4 (SEQ IDNO:1 or amino acids 1 to 38 of SEQ ID NO:1).
 10. The bispecific proteincomplex according to claim 9, wherein the scFv is 52SR4 (SEQ ID NO:3 oramino acids 1 to 243 of SEQ ID NO:3).
 11. The bispecific protein complexaccording to claim 8, wherein Y is a peptide.
 12. The bispecific proteincomplex according to claim 7, wherein X is a peptide.
 13. (canceled) 14.The bispecific protein complex according to claim 12, wherein Y is a Fabfragment, a Fab′ fragment, a scFv or a sdAb.
 15. The bispecific proteincomplex according to claim 14, wherein: a. the Fab fragment, the Fab′fragment, the scFV or the sdAb is specific to the peptide GCN4 (SEQ IDNO:1 or amino acids 1 to 38 of SEQ ID NO:1) or b. Y is a Fab fragment, aFab′ fragment, a scFV, or sdAb, or c. Y is a Fab fragment, or d. Y is ascFV or sdAb, or e. A is a scFV or sdAb. 16-19. (canceled)
 20. Thebispecific protein complex according to claim 15, wherein: a. X isindependently selected from a Fab fragment, a Fab′ fragment, a scFv, asdAb and an antigen with the proviso that when X is a peptide Y is a Fabfragment, a Fab′ fragment, a scFv or a sdAb and when X is a Fabfragment, a Fab′ fragment, a scFv or a sdAb then Y is an antigen or b. Yis independently selected from Fab fragment, a Fab′ fragment, a scFv, asdAb and an antigen with the proviso that when Y is a peptide X is a Fabfragment, a Fab′ fragment, a scFv, a sdAb and when Y is a Fab fragment,a Fab′ fragment, a scFv or a sdAb then X is an antigen.
 21. (canceled)22. The bispecific protein complex according to claim 20, wherein: a. Yis a Fab, or b. Y is a scFV or sdAb.
 23. (canceled)
 24. The bispecificprotein complex according to claim 20, wherein the Fab fragment, theFab′ fragment, the scFv or the sdAb is specific to the peptide GCN4 (SEQID NO:1 or amino acids 1 to 38 of SEQ ID NO:1).
 25. The bispecificprotein complex according to claim 24, wherein the scFv is 52SR4 (SEQ IDNO:3 or amino acids 1 to 243 of SEQ ID NO:3).
 26. The bispecific proteincomplex according to claim 20, wherein X or Y is a peptide. 27.(canceled)
 28. The bispecific protein complex according to claim 1 whichcomprises no more than two scFvs.
 29. The bispecific protein complexaccording to claim 1, wherein A and/or B is specific for an antigenselected from the group comprising: cell surface receptors,co-stimulatory molecules, checkpoint inhibitors, natural killer cellreceptors, Immunoglobulin receptors, TNFR family receptors, B7 familyreceptors, adhesion molecules, integrins, cytokine/chemokine receptors,GPCRs, growth factor receptors, kinase receptors, tissue-specificantigens, cancer antigens, pathogen recognition receptors, complementreceptors, hormone receptors or soluble molecules.
 30. A compositioncomprising one or more bispecific protein complexes defined in claim 1.31-32. (canceled)
 33. A method for detecting synergistic biologicalfunction in a heterodimerically-tethered bispecific protein complex offormula A-X:Y-B, wherein: A-X is a first fusion protein; Y-B is a secondfusion protein; X:Y is a heterodimeric-tether; : is a bindinginteraction between X and Y; A is a first protein component of thebispecific protein complex independently selected from the groupcomprising a Fab fragment, a Fab′ fragment, sdAb and a single chain Fv(scFv); B is a single chain Fv or sdAb; X is a first binding partner ofa binding pair independently selected from an antigen, a Fab fragment, aFab′ fragment, a single chain Fv and sdAb; and Y is a second bindingpartner of the binding pair independently selected from antigen, a Fabfragment, a Fab′ fragment, a single chain Fv and a sdAb; with theproviso that when X is an antigen Y is a Fab fragment, a Fab′ fragment,a single chain Fv or a sdAb specific to the antigen represented by X andwhen Y is an antigen X is a Fab fragment, a Fab′ fragment, a singlechain Fv or a sdAb specific to the antigen represented by Y, said methodcomprising the steps of: (i) testing for activity in a functional assayfor part or all of a multiplex comprising at least oneheterodimerically-tethered bispecific protein complex; and (ii)analysing the readout(s) from the functional assay to detect synergisticbiological function in the heterodimerically-tethered bispecific proteincomplex.
 34. (canceled)
 35. The method according to claim 33, wherein:a. the multiplex comprises at least two heterodimerically-tetheredbispecific protein complexes or b. the heterodimerically tetheredbispecific protein complex(es) do not contain an Fc region or c. theheterodimerically tethered bispecific protein complexes are not purifiedprior to testing or d. the A-X and Y-B fusion proteins are expressedtransiently and not purified before being mixed in a 1:1 molar ratio togenerate each heterodimerically tethered bispecific protein complex.36-52. (canceled)